Membrane stacks

ABSTRACT

The present application discloses a membrane stack comprising a first and second membrane layers, and a spacer layer disposed between said first and second membrane layers, said membrane stack configured such that fluid passes through said membrane stack in a direction substantially perpendicular to the plane of said membrane layers and said spacer layer. The application also discloses a module comprising a membrane as described above, said module having a fluid flow path that is substantially perpendicular to the plane of the major surface of the membrane and spacer layers in said membrane stack.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a National Stage filing of International ApplicationPCT/CA2005/000518, filed Apr. 7, 2005, entitled “MEMBRANE STACKS”,claiming priority to and the benefit of U.S. Provisional PatentApplication No. 60/560,027, filed Apr. 8, 2004. Both references areexpressly incorporated by reference herein, in their entirety.

FIELD OF THE INVENTION

The invention relates to filtration membranes, particularly stacks ofmembrane layers separated by spacer layers.

BACKGROUND OF THE INVENTION

A new generation of membranes suited for membrane chromatography and avariety of other applications including microfiltration,ultrafiltration, membrane absorbers, etc. has been developed. Themembranes are useful in e.g. bio-separations where they can be used asdisposable devices that have superior performance to existing separationsystems.

These membranes comprise a macroporous gel anchored within a suitablenon-woven support. In order to achieve high protein and other moleculeadsorption capacities in chromatography and related applications, thegels are typically relatively soft, thereby allowing diffusion of thetarget molecules into the gel phase. Using this approach, dynamiccapacities that are a factor of six higher than the best alternativemembrane products can be achieved.

There is a downside to using soft macroporous gels. This comes from thefluxes that can be achieved with these membranes, particularly when themembranes are combined to form a stack as is typically used in membranechromatography applications. Ideally, the flux drop at a given pressureshould decrease linearly as additional membranes are added to a stack.It is well known that the flux through a membrane is inverselyproportional to the thickness of the active layer. For example, whereasthe flux of a single Q-type membrane at 100 kPa is 2500 kg/m²h wheneight layers are combined, the resulting flux is 135 kg/m²h, rather thanthe expected value of 2,500/8=312 kg/m²h. This reduction in flux can beoffset by increasing the trans-membrane pressure. But this comes withthe added cost of more expensive membrane housings, pumps, etc.

The problem becomes greater in higher capacity gel based membranes withsofter gels. Stacks of these membranes exhibit a non-linear pressureflux relationship. In severe cases, the flux reaches a limiting value asthe pressure is increased. This effect limits the thickness of themembrane stacks that can be used if high flow rates are to bemaintained.

It is known to use certain types of spacers between layers in membranestacks for particular purposes.

Spacers are common elements in membrane systems or modules used indesalination processes such as reverse osmosis, electrodialysis,electro-deionization and diffusion dialysis. In these systems a “spacer”is a device that provides a generally defined distance between twoadjacent membrane sheets to allow cross-flow of a liquid between the twomembrane sheets. A feed fluid spacer is a porous spacer layer thatprovides for the passage of the feed fluid over and parallel to theactive side of a membrane. This flow parallel to a membrane surface istermed cross-flow. The feed fluid spacer serves the function ofdirecting the feed fluid to cover the active side of the membrane in auniform manner. The spacer may also impart turbulence to the feed fluidto provide good mixing in the feed fluid as it travels over the activeside of the membrane and to reduce concentration polarization.

A typical spiral wound membrane module comprising such spacers is shownin FIG. 1.

The spiral module contains tightly packed membranes sandwiched betweenmesh spacers and wrapped around a small-diameter central tube. There aretwo spacers in this module; a permeate carrier layer as well as a feedspacer/distributor.

The membrane stack includes two, long semipermeable membranes with aspacer mesh between them. This is then wound up in a spiral tube withanother spacer, the permeate carrier layer, to separate the outer,permeate sides of the stack. The mesh separator or spacer on the feedside allows the feed to be forced in one side of the spiral cylinder andout the other side. Pressure on the feed side forces some of the water(typically less than 15% of the feed water passes through the membraneon a single pass) to pass through the membrane where it is collected inthe space between the membranes. The resulting permeate then flowsaround the spiral where it is collected in the centre of the tube. Anearly description of a spiral wound device is given in Bray D. T.Reverse osmosis purification apparatus. U.S. Pat. No. 3,417,870. 1968.

The key point in this design of a membrane module is that the spacers onthe feed and permeate side allow flow of the two streams parallel to thesurfaces of the membranes.

Another common example of membrane stacks is in electrodialysis cells.Electrodialysis is a process that uses a direct electrical current toremove salt, other organic constituents, and certain low molecularweight organics from brackish water. With this technique several hundredflat, ion permeable membranes and water flow spacers are verticallyassembled in a stack. Half of the membranes allow positively chargedions, or cations, to pass through them. The other half-anion-permeablemembranes—allow negatively charged ions to pass through them. The anionpermeable membranes are alternately placed between the cation-permeablemembranes. Each membrane is separated from the adjacent membrane in thestack by a polyethylene flow spacer.

An electrical current is established across the stack by electrodespositioned at both ends of the stack. Brackish water is pumped at lowpressures into the flow spacers between each membrane and passes throughthe cell exiting on the opposite side. While passing through the cell,ionic constituents are electrochemically driven through the membranes oneither side of the channel. This results in removal of salts from thebrackish water stream and the formation of a concentrated salt steam inthe intervening channels. The spacers serve to hold the membranes inplace and allow the flow of the brackish water and resulting moreconcentrated streams parallel to the membrane surfaces. Typical stackdesigns are shown in the patents of Iaconelli (U.S. Pat. No. 3,695,444)and Olsen (U.S. Pat. No. 3,623,610).

It will be appreciated from the above that spacers are conventionallyused to permit fluid to flow in the plane of the major surface of amembrane. The spacers used in such devices as spiral wound modules wheretangential flow occurs across-the surface of the membranes from a feedchannel containing the spacer, are typically quite thick and constructedsuch that flow channels remain even when sandwiched tightly between twomembranes. This flow path is constructed to be tortuous such thatmovement of the feed through the spacer is turbulent. The design ofthese spacers is complex with different sized elements making up themesh like material so as to provide the tortuous path.

But the art does not disclose the use of spacers in instances where thefluid flow path is substantially perpendicular to the plane of a majorsurface of the membrane layers in a membrane stack.

BRIEF SUMMARY

We have found that, by inserting spacer layers between individualmembrane layers, the flux of the membrane stack can be increaseddramatically. Surprisingly, the interleaving of spacer layers betweenthe membrane layers in a membrane stack did not lead to a loss ofresolution of the stacks in a separation. In fact, the breakthroughcurve of a four-membrane stack with interleaving spacer layers was foundto be somewhat sharper than the four layers without interleaving. Thus,the invention thus not only improves substantially the flux of a stackat a given pressure but also improves its performance in a separation.

Accordingly, in one aspect, the invention provides a membrane stack,comprising first and second membrane layers; and a spacer layer disposedbetween the first and second membrane layers; the membrane stackconfigured such that fluid passes through the membrane stack in adirection substantially perpendicular to the plane of the membranelayers and the spacer layer.

In another aspect, the invention provides a module comprising a membranestack as described above, the module having a fluid flow path that issubstantially perpendicular to the plane of the major surface of themembrane and spacer layers in said membrane stack.

In another aspect, the invention provides a method for separating asubstance from a fluid, comprising the step of passing the fluid througha membrane stack or module as described above.

DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1: Illustration of the construction of a spiral-membrane reverseosmosis module, (prior art).

FIGS. 2 and 3: A schematic diagram of a stack of membranes with spacers.

FIG. 4: Schematic diagram of Empore 96 well plate format (4A) withdetails of the pre-filter assembly (4B).

FIG. 5: A schematic view of a stack of six membranes with threedifferent types of properties. The membranes are separated by spacerlayers.

FIG. 6: Water flux versus pressure through 4-layers of a high capacityQ-type membrane with and without spacers between the layers.

FIG. 7: Effect of increase in a number of layers on a water flux for amembrane stack with and without spacers.

FIG. 8: Effect of spacers on the sharpness breakthrough BSA adsorptioncurve for 4-layers Q-type of membrane.

FIG. 9: Environmental scanning electron microscope (ESEM) image of amacroporous poly(APTAC) gel.

FIG. 10: ESEM image of a macroporous poly(APTAC) gel incorporated into asupport member in the form of a membrane.

FIG. 11: Lysozyme adsorption curve of the membrane prepared in Example6, below. The membrane volume is 0.467 ml.

FIG. 12: BSA adsorption curve of the membrane prepared in Example 11.

FIG. 13: Graphical representation of the hydraulic radius as a functionof mass gain with photo- and thermally initiated porous gel containingmembranes. Gel: poly(glycidyl methacrylate-co-ethylene diacrylate);solvents: dodecanol (DDC)/cyclohexanol (CHX) 9/91.

FIG. 14: Graphical representation of the mass gain as a function oftotal monomer concentration during the preparation of compositemembranes.

FIG. 15: AFM image of the surface of the AM610 membrane; (scanned area:100 μm²).

FIGS. 16A and 16B: ESEM images of a nascent (FIG. 16A) and AM610 (FIG.16B) surfaces; (magnification: 5000×).

FIGS. 17A and 17B: ESEM images of the surface of AM611 membrane;(magnification: FIG. 17A—5000×, FIG. 17B—3500×).

FIGS. 18A and 18B: ESEM images of membranes AM610 (FIG. 18A) and AM611(FIG. 18B); (magnification 5000×).

FIG. 19: Lysozyme adsorption curve of the membrane prepared in Example18. The membrane volume is 0.501 ml.

FIG. 20: Lysozyme adsorption curve of the membrane prepared in Example19. The membrane volume is 0.470 ml.

FIG. 21: Lysozyme adsorption curve of the membrane prepared in Example20. The membrane volume is 0.470 ml.

FIG. 22: ESEM image of a wet macroporous gel that is the product ofExample 28.

FIG. 23: ESEM image of a wet microporous gel in a fibrous non-wovensupport member that is the product of Example 29.

FIG. 24: Graphical results of using a multi-membrane stack ofmacroporous gel-filled membranes of Example 25 in a protein (BSA)adsorption test.

FIG. 25: Graphical display of the effect of monomer concentration on themass gain of macroporous gel-filled membranes.

FIG. 26: Graphical display of the effect of ionic interactions on theflux through a macroporous gel-filled membrane at a pressure of 100 kPa.

FIG. 27: Graphical display of the changes in trans-membrane pressure andpermeate conductivity as a function of salt concentration in thepermeate (A and B) and the changes of trans-membrane pressure as afunction of permeate conductivity (salt concentration)(C).

FIG. 28: The relationship between trans-membrane pressure, conductivityand absorbance for the HIgG ultrafiltration carried out in Example 42.

FIG. 29: The relationship between conductivity and absorbance for theHSA ultrafiltration carried out in Example 42.

FIG. 30: The relationship between trans-membrane pressure, conductivityand absorbance for the HSA/HIgG ultrafiltration carried out in Example42.

DETAILED DESCRIPTION

Membrane Layers

The membrane layers may be any kind of filtration membrane layers, andthe invention is not limited to the use of particular types of membranelayers or combinations thereof.

Preferred membrane layers include those comprising structured gelsincorporated into a support, as described in U.S. Pat. No. 6,258,276(Childs et al.); International Publication No. WO 03/08078 (Childs etal.), R. F. Childs, A. M. Mika, The design of high performance,gel-filled nanofiltration membranes, in A New Insights into MembraneScience and Technology: Polymeric, Inorganic and BiofunctionalMembranes@ Elsevier, Edit. A. Butterfield and D Bhattacharyya, 2003 pp353-375; and U.S. Pat. No. 5,160,627 (Cussler et al.), all of which areincorporated herein by reference. In a particularly preferredembodiment, the membrane layers include macroporous gel-filled membranesas described herein and in International Publication No. WO 2004/073843(Childs et al.), incorporated herein by reference. These structured gelmembranes (MGel membranes) have significant advantages over existingtraditional membranes. In terms of UF/MF applications they have verynarrow pore-size distributions, have the ability to have dynamicpore-size control, are exceptionally hydrophilic, exhibit much lowerfouling than other membranes and can be made at very low cost. In termsof adsorption/specific binding membranes the MGel platform providesmembranes with exceptionally high loading capacities, the readycapability of introduction of specific ligands, and the ability to makesingle membranes with much higher thicknesses than conventionalmembranes. This latter feature means that fewer layers have to be usedto obtain a given total stack thickness. Other useful membrane layersinclude, without limitation, microfiltration membranes orultrafiltration membranes as are known in the art.

In one embodiment, a gel-filled membrane is used in combination with aconventional membrane such as a microfiltration or ultrafiltrationmembrane as mentioned above.

In another embodiment, a conventional microfiltration or ultrafiltrationmembrane is used in combination with, for example, adsorptive typemacroporous gel based membranes. Alternatively, a macroporous gel basedsize exclusion membrane (microfiltration or ultrafiltration) may becoupled with an affinity type membrane(s). In each case a spacer may beused between the conventional membrane and the gel-filled membrane (aswell as between any two or more gel filled membranes forming the stack).

In another embodiment, the membrane stack comprises macroporous gelbased membranes with the same functionality but whose pore-size ordensity of active is or can be different in each layer. This combinationmay be used in e.g. a tubular arrangement as described herein, so as toeven out hydraulic flow through the pores as the radius of the tubeincreases.

In another embodiment, homogeneous gel-filled membranes are used. Theseare effective as very tight ultrafiltration membranes and also asnanofiltration type membranes. In one embodiment, at least one suchmembrane is used in combination with a series of adsorptive typemacroporous gel based membranes.

Spacers

Without intending to be bound to any particular theory, it is believedthat the spacer layer serves largely to eliminate compression of surfacelayers, particularly on the macroporous gel based membranes. Themacroporous gels are quite soft, mechanically weak and easily deformed.In order to use the macroporous gels as an effective separationmembrane, it has been discovered that their mechanical properties couldbe increased by incorporating the gel into a porous membrane ormaterial, including non-woven fabrics. Inevitably, such membranes willhave a surface layer of the gel that is not strengthened by the support.Under conditions of hydraulic flow under pressure, with two membranesput together, it is believed that there is a compression of theunsupported gel surface layers. This compression will lead to areduction in effective pore-size in this compressed region and acommensurate drop in flux over what would have been expected.Introducing a spacer layer between the two membranes would appear toprevent this compression of the surface gel layers.

The spacer may be any material that can separate the membrane layers andpermit hydraulic flow of fluid substantially perpendicularly to themajor surfaces of the membrane layers. It is desirable that the spacersdo not permit fluid flow tangential to the major membrane surfaces.However, tangential fluid flow may occur across the surface of the firstmembrane in a stack.

Spacers may desirably possess one or more of the following features orproperties:

1. low non-specific binding for proteins or other biomolecules in thosecases where the stack is to be used for biomolecule processing;

2. contain low or very low amounts of leachable materials;

3. a thickness in the range of preferably about 50 μm microns to about500 μm;

4. be formed of a mesh material having a high porosity with a mesh size(i.e. the size of openings in the mesh) of about 50 μm to 5,000 μm,preferably 500 μm to about 1000 μm.

By way of non-limiting examples, the spacer layer may be a material thatis woven, moulded, formed, extruded, knitted, cast or formed in place.

In one embodiment, a thin mesh-like material having uniform thickness ofthe elements making up the mesh is used. Such a spacer can fit snuglybetween two membranes and only permit hydraulic flow to occurperpendicularly to the surfaces of the membranes.

Examples of commercially available spacer layers include symmetricalfiltration netting made of polypropylene of thickness of 500 μm and meshopening of 1000 μm (Naltex, Austin, Tex., USA) and polyester mesh ofthickness of 350 μm and mesh opening of 500 μm (Polyester Monofilament)Great Lakes Filters Inc. Hillsdale Mich.

Membrane Stacks

Membrane stacks comprise a plurality of layers, including at least onefiltration membrane layer and at least one spacer layer. The terms“membrane stack” and “layer” are used with reference to the fluid paththrough the membrane stack. As shown in FIGS. 2 and 3, the fluid paththrough the membrane stacks of the invention is substantiallyperpendicular to the plane of the major surfaces of the layers in thestack. This is in contrast to some types of membrane systems, whereinfluid flow is largely in or parallel to the plane of the layers in astack. Hence, in the instant invention, the terms “membrane stack” and“layer” principally describe the arrangement of filtration membranes andspacers relative to the fluid path.

In some membrane stacks, the layers will indeed be composed of separate,discrete sheets of membrane material and spacer material. Such anarrangement is depicted in FIGS. 2 and 3. In other instances, such as inthe case of a spiral wound device, a single sheet of membrane materialor spacer material may define multiple layers in the membrane stack. Thefluid path crossing the same sheet of material multiple times in thespiral wound roll. In this embodiment, the sheet of membrane or spacermaterial constitutes a “layer” each time it crosses the fluid path andthe “membrane stack” describes the organization and arrangement of thelayers. Such embodiments are distinguished from the spiral type deviceshown in FIG. 1, because in the device shown in FIG. 1, fluid flow is inthe plane of the layers, and not perpendicular to it. In contrast, werethe fluid instead to pass into the core of the spiral, and then flowradially outwardly, the fluid flow would be substantially perpendicularto the plane of the membrane and spacer layers.

Membrane stacks will typically comprise a plurality of filtrationmembrane layers interleaved with spacer layers, such that each adjacentpair of membrane layers is separated by a spacer layer. However,particularly because the membrane stack may contain other types oflayers, or filtration membrane layers that are not especiallysusceptible to compression, in some embodiments, not every adjacent pairof filtration membrane layers will be separated by a spacer layer.Spacer layers may be used selectively, and included in the membranestack only where needed to separate membrane layers susceptible to beingcompressed together.

Non-limiting examples of various possible combinations of membranelayers in the membrane stack are set forth in Table 1 below.

Membrane stacks will usually, although not necessarily, comprise atleast one macroporous gel-filled membrane layer as described herein.Some embodiments may involve a combination of one or more macroporousgel-filled membrane layers coupled with one or more conventionalmembrane layers.

In one embodiment, conventional microfiltration and/or ultrafiltrationmembrane layers are used in combination with, for example, adsorptivetype macroporous gel based membrane layers. Conversely, a macroporousgel based size exclusion membrane layer (microfiltration orultrafiltration) may be used with one or more affinity type conventionalmembrane layers. In each case, a spacer layer would be used between theconventional membrane and the gel-filled membrane layer (as well asbetween any two or more gel filled membrane layers forming the stack).

In another embodiment, the membrane stack comprises one or moremacroporous gel based membranes with the same functionality but whosepore-size or density of active material may be different in each layer.This may be useful in, for example, tubular arrangements so as to evenout hydraulic flow through the pores as the radius of the tubeincreases.

In another embodiment, one or more membrane layers are homogeneousgel-filled membrane layers, as defined in R. F. Childs, A. M. Mika, Thedesign of high performance, gel-filled nanofiltration membranes. A NewInsights into Membrane Science and Technology: Polymeric, Inorganic andBiofunctional Membranes@ Elsevier, Edit. A. Butterfield and DBhattacharyya, 2003 pp 353-375; U.S. Pat. No. 6,258,276 (Childs et al.);International Publication No. WO 03/08078 (Childs et al.); and U.S. Pat.No. 5,160,627 (Cussler et al.), the relevant parts of which areincorporated herein by reference. These are useful as very tightultrafiltration membranes and also as nanofiltration type membranes.They exhibit the same type of surface gel layers as macroporousgel-filled membrane layers described below and the spacer layers can beeffective where stacks are formed. Such homogeneous gel-filled membranelayers may be used in combination with a series of adsorptive typemacroporous gel based membrane layers.

In one embodiment, the membrane stack is a multifunction membrane stackwherein several membranes are placed one on top of another to provide acomposite separation device. These generally comprise a stack offilters/membranes that have size selection properties in the upperlayers and adsorption functionality in the bottom layers as depicted inFIG. 4. A typical example is found with the 3M Empore™ product line.FIG. 5 is a schematic depiction of a stack of six membranes with threedifferent types of properties (2, 4, 6) separated by spacer layers 8.

Modules Comprising Membrane Stacks

Membrane stacks are generally assembled into a module for use. Themodule contains or carries the membrane stack and is configured suchthat the fluid is directed through the membrane stack in a fluid paththat is substantially perpendicular to the plane of the major surfacesof the layers in the membrane stack. Generally, the module constrainsthe fluid such that all or a desired portion of the fluid flow must passthrough the membrane stack, i.e. not escape around it.

Modules can be e.g. of the spiral wound, plate and frame, tubular, andhollow-fibre type arrangements (Mulder M. Basic principles of membranetechnology. Kluwers Academic Publishers (1996) p. 564, and Baker R. W.Membrane technology and applications. New York: McGraw-Hill, (2000) p.514.) The initial membrane module configurations were of a plate andframe type. This configuration was based on designs in the filtrationindustry, U.S. Pat. Nos. 3,473,668 and 3,209,915.

In one embodiment, the membrane stack is integrally moulded such thatthe spacers and the membranes are a single unit that are joined to afeed/distributor system and permeate collection system with flow aroundthe stack being prevented. This type of configuration may take the formof e.g. a flat disk.

In another embodiment, fluid flow around the membrane stack is preventedthrough the use of a sealing device or sealing means such as pressure onthe outside edges of the stack.

In another embodiment, the module comprises a membrane stack configuredas a series of touching, concentric rings forming a tubular device. Flowmay be from the centre of the tube through the walls of the tube to theoutside or the reverse. This type of tubular device does not have to bea strict cylinder. Pleating or other types of folding may be introducedsuch that the surface area and membrane stack volume can be increased inthe module.

The above types of arrangements are illustrated generically in FIG. 3.Blocks 10 represent the membrane, hatched blocks 12 represent the spacerlayers. The solid blocks 14 at each side of the stack represent a sealthat prevents flow around the membrane stack. In these embodiments, flowthrough the stack is through the membrane layers. The spacer layersenable the flow to be channelled from one membrane layer to the next inthe stack with minimal mixing across the surface of the membrane layers.The fluid feed may be supplied in either a tangential 16 or flow through18 mode, but once the feed enters the stack then it will flow as shownin a direction 20 that is substantially perpendicular to the stack. Itis preferable that the feed is evenly distributed over the entire feedsurface of the membrane layers.

In another embodiment, where a significant number of layers is desired,the module may take the form of a tubular device such as spiral woundarrangement in which a membrane layer and a spacer layer are woundconcentrically. The centre of the resulting tube thus forms one flowchannel (feed or permeate), and the outside of the tube forms the secondflow channel. In these tubular devices, the ends of the membrane stackmay be sealed such that flow has to take place through the stack.

TABLE 1 Layer 1 Layer 2 Layer 3 Membrane use Type Function Type FunctionType Function Examples Neutral UF Size Strong or Adsorption/release — —One step primary recovery of proteins from or MF separation weak basechromatography fermentation broths Neutral UF Size Strong orAdsorption/release — — One step primary recovery of proteins from or MFseparation weak acid chromatography fermentation broths Charged Chargeaided Strong or Adsorption/release — — One step primary recovery ofproteins from UF or MF size weak base chromatography fermentation brothsseparation Charged Charge aided Strong or Adsorption/release — — Onestep primary recovery of proteins from UF or MF size weak acidchromatography fermentation broths separation Neutral UF Size SpecificAdsorption/release — — One step primary recovery of proteins from or MFseparation binding chromatography fermentation broths Neutral UF SizeSpecific Adsorption/release — — One step primary recovery of proteinsfrom or MF separation binding chromatography fermentation broths ChargedCharge aided Specific Adsorption/release — — One step primary recoveryof proteins from UF or MF size binding chromatography fermentationbroths separation Charged Charge aided Specific Adsorption/release — —One step primary recovery of proteins from UF or MF size bindingchromatography fermentation broths separation All of the abovevariations/combinations Covalently Protein Separation, concentration anddigestion of bound hydrolysis - proteins for analysis sequencing Trypsindigestion All of layer 1 Covalently Protein hydrolysis - Separation,concentration and digestion of combinations bound digestion proteins foranalysis sequencing Trypsin All of layer 1 Covalently Protein hydrolysisVariable Control Separation, concentration and digestion of combinationsbound digestion pore-size release of proteins for analysis sequencingTrypsin peptides All of layer 1 Covalently Protein hydrolysis UF AllowSeparation, concentration and digestion of combinations bound digestionmembrane peptide proteins for analysis sequencing Trypsin release butprevent undigested protein releaseUses of Membrane Stacks and Modules

The membrane stacks and modules of the invention may be used in allapplications where filtration membranes or modules are used. Theseinclude, for instance, separation and/or reaction/separationapplications. They may also be used for e.g. analytical devices such asmulti-well plates or in large scale therapeutic material production.

In one embodiment, the membrane stacks are used for membranechromatography applications, particularly in the large scale separationand production of bio-molecules. These include the isolation andpurification of antibodies, therapeutic proteins, plasmids, etc.Disposable membrane stacks offer significant advantages over the moreconventional resin bead chromatography in terms of performance,regulatory compliance and process economics.

In addition to chromatography stacks consisting of a single membranetype, the invention can also greatly improve the performance ofmulti-element stacks in which membranes of different properties arecombined into a single stack. These multi-element stacks allow for asimplification of the bioprocess purification train coupled withenhanced recoveries of the target biomolecules. This may translate intosignificant process cost savings.

Macroporous Gel-Filled Membranes

As discussed above, the membrane stack of the invention can, in someembodiments, comprise macroporous gel-filled membranes. Macroporousgel-filled membranes can be defined as composite materials that comprisea support member that has a plurality of pores extending through thesupport member and, located in the pores of the support member andessentially filling the pores of the support member, a macroporouscross-linked gel. In some embodiments, the macroporous gel used isresponsive to environmental conditions, providing a responsive membrane.

The macroporous gel fills the pores of the support laterally, i.e.substantially perpendicular to the direction of the flow through themembrane. By “fill” we mean that, in use, essentially all liquid thatpasses through the membrane must pass through the macroporous gel. Asupport member whose pores contain macroporous gel to such an amountthat this condition is satisfied is regarded as filled. Provided thatthe condition is met that the liquid passes through the macroporous gel,it is not necessary that the void volume of the support member becompletely occupied by the macroporous gel.

The porous support member, or host, may be hydrophilic or hydrophobicand can be, for example, in the form of a membrane, a chromatographybed, or a filtration bed. The support member provides the mechanicalstrength to support the macroporous gel. The macroporous gel provides alow resistance to hydraulic flow, enabling high flow rates to beachieved with low reductions in pressure across the macroporousgel-filled membrane. The macroporous gel also provides the separatingfunction of the membrane in chromatographic and filtration applications.

A gel is a cross-linked polymer network swollen in a liquid medium. Theswelling liquid prevents the polymer network from collapsing and thenetwork, in turn, retains the liquid.

Gels are typically obtained by polymerization of a monomer and apolyfunctional compound (a cross-linker), or by cross-linking across-linkable polymer, in a solvent which is a good solvent for theformed polymer network and which swells the polymer network. The polymerchains in such a network can be assumed to be uniformly distributedthroughout the whole volume of the network and the average distancebetween the chains, known as mesh size, is determined by thecross-linking density. As the concentration of the cross-linker isincreased, the density of cross-links in the gel also increases, whichleads to a smaller mesh size in the gel. The smaller mesh size resultsin a higher resistance to the flow of liquids through the gel. As theconcentration of the cross-linker is increased further, the constituentsof the gel begin to aggregate, which produces regions of high polymerdensity and regions of low polymer density in the gel. Such gels exhibitwhat has been called microheterogeneity. This aggregation normallycauses the gel to display a higher permeability to liquids, as the flowof liquids takes place primarily through the areas in the gel that havea lower polymer density. The low density areas of the gels are definedas draining regions while the higher density aggregates are callednon-draining regions. As the concentration of the cross-linker isincreased even further, leading to more cross-links, the gel can developregions in which there is essentially no polymer. These regions arereferred to as “macropores” in the present specification.

It is possible to compare the hydrodynamic (Darcy) permeability of aparticular membrane with a reference material. The reference material isobtained by filling the pores of a support member identical with that ofthe macroporous gel-filled membrane with a homogeneous gel ofessentially the same chemical composition and the similar mass as thegel of the macroporous gel-filled membrane, that is a gel composed ofthe same monomers formed in a good solvent, but cross-linked only tosuch an extent that the gel remains homogeneous and aggregation intoregions of high and low polymer density does not occur. Membranescomprising macroporous gels display hydrodynamic (Darcy) permeabilitiesthat are at least one order of magnitude higher than those of thecorresponding reference materials, and in some instances thepermeabilities are more than two or even more than three orders ofmagnitude higher. In this specification, a macroporous gel-filledmembrane whose hydrodynamic (Darcy) permeability is at least an order ofmagnitude greater than that of the corresponding reference material issaid to have a permeability ratio greater than 10.

The permeability ratio is closely related to the size of the macroporesin the macroporous gel-filled membrane. For size-exclusion separationssuch as ultrafiltration, the permeability ratio can be fairly close to10. In other applications, for example adsorption, synthesis or cellgrowth, where larger macropores are used, the permeability ratio canreach, in some embodiments, values of 100 or greater, or even 1000 orgreater. In some instances it is possible to calculate the hydrodynamicpermeability of homogeneous gels, in accordance with the teachings ofMika A. M. and Childs R. F., Calculation of the hydrodynamicpermeability of gels and gel-filled microporous membranes, Ind. Eng.Chem. Res., vol. 40 (2001), p. 1694-1705, incorporated herein byreference. This depends upon data for the particular gel polymer beingavailable.

From the hydrodynamic permeability there can be derived the hydrodynamicradius, defined as the ratio of the pore volume to the pore wettedsurface area. It can be calculated from the hydrodynamic (Darcy)permeability using the Carman-Kozeny equation as given, for example, inthe book J. Happel and H. Brenner, Low Reynolds Numbers Hydrodynamics,Noordhof of Int. Publ., Leyden, 1973, p. 393, incorporated by referenceherein. It is necessary to assume a value for the Kozeny constant andfor the purpose of these calculations the inventors assume a value of 5.Macroporous gel-filled membranes are found to have a hydrodynamic radiusmore than three times as high as the hydrodynamic radius of thecorresponding reference material.

From the definition of the hydrodynamic permeability it can be derivedthat two macroporous gel-filled membranes of the same thickness willhave hydrodynamic fluxes at the same pressure that will have the sameratio as their permeability ratio.

The size of macropores in the gel can be within a broad range, from afew nanometers to several hundred nanometers. Preferably, the porous gelconstituent of the macroporous gel-filled membrane has macropores ofaverage size between about 10 and about 3000 nm, has volume porositybetween 30 and 80% and a thickness equal to that of the porous supportmember. In some embodiments, the average size of the macropores ispreferably between 25 and 1500 nm, more preferably between 50 and 1000nm, and most preferably the average size of the macropores is about 700nm.

In the absence of a support member, the macroporous gels may be non-selfsupporting, and they may change or even lose their porosity when dried.By inserting the macroporous gel within a porous support member,mechanical strength is conferred upon the macroporous gel. Theutilization of macroporous gels creates a macroporous gel-filledmembrane that permits larger molecules, such as biological molecules, toenter the macropores and the solution containing such molecules totraverse the gel at a high flux.

By a “responsive macroporous gel-filled membrane” is meant a macroporousgel-filled membrane which comprises a macroporous gel whose pore-sizecan be controlled by varying specific environmental conditions.

General Characteristics of the Macroporous Gel-Filled Membrane

Preferably, the macroporous gel is anchored within the support member.The term “anchored” is intended to mean that the gel is held within thepores of the support member, but the term is not necessarily restrictedto mean that the gel is chemically bound to the pores of the supportmember. The gel can be held by the physical constraint imposed upon itby enmeshing and intertwining with structural elements of the host,without actually being chemically grafted to the host or support member,although in some embodiments, the macroporous gel may become grafted tothe surface of the pores of the support member.

It will be appreciated that as the macropores are present in the gelthat fills the pores of the support member, the macropores must besmaller than the pores of the support member. Consequently, the flowcharacteristics and separation characteristics of the macroporousgel-filled membrane are dependent on the characteristics of themacroporous gel, but are largely independent of the characteristics ofthe porous support member, with the proviso, of course, that the size ofthe pores present in the support member is greater than the size of themacropores of the gel. The porosity of the macroporous gel-filledmembrane can be tailored by filling the support member with a gel whoseporosity is primarily controlled by the nature and amounts of monomer orpolymer, cross-linking agent, reaction solvent, and porogen, if used. Aspores of the support member are filled with the same macroporous gelmaterial, there is achieved a high degree of consistency in propertiesof the macroporous gel-filled membrane, and for a particular supportmember these properties are determined largely, if not entirely, by theproperties of the macroporous gel. The net result is that the inventionprovides control over macropore-size, permeability and surface area ofthe macroporous gel-filled membranes.

The number of macropores in the macroporous gel-filled membrane is notdictated by the number of pores in the support material. The number ofmacropores in the macroporous gel-filled membrane can be much greaterthan the number of pores in the support member, although the macroporesare smaller than the pores in the support member. As mentioned above,the effect of the pore-size of the support material on the pore-size ofthe macroporous gel is generally quite negligible. An exception to thisis found in those cases where the support member has a large differencein pore-size and pore-size distribution, and where a macroporous gelhaving very small pore-sizes and a narrow range in pore-sizedistribution is sought. In these cases, large variations in thepore-size distribution of the support member are weakly reflected in thepore-size distribution of the macroporous gel. As such it is preferableto use a support member with a somewhat narrow pore-size range in thesesituations.

The properties of the macroporous gel-filled membrane can be tuned, byadjusting the average pore diameter of the macroporous gel. For somepurposes, for example ultrafiltration by means of size exclusion, smallpores may be required. For other purposes, for example use as a solidsupport for a chemical synthesis involving fast-kinetics, large poresmay be required. The size of the macropores is mainly dependent on thenature and concentration of the cross-linking agent, the nature or thesolvent or solvents in which the gel is formed, the amount of anypolymerization initiator or catalyst and, if present, the nature andconcentration of porogen.

Generally, as the concentration of cross-linking agent is increased, thesize of the macropores in the gel is also increased. For example, themolar ratio of polyfunctional compound(s)(cross-linking agent) tomonomer(s) may be in the range of from about 5:95 to about 70:30,preferably in the range of from about 10:90 to about 50:50, and morepreferably in the range of from about 15:85 to about 45:55.

The components of the macroporous gel are introduced into the pores ofthe support member by means of a liquid vehicle, and solvent selectionfor in situ polymerization or cross-linking plays a role in obtainingporous gels. Generally, the solvent or solvent mixture should dissolvemonomers and polyfunctional compounds, or cross-linkable polymers andcross-linking agents, over a wide range of concentrations. If thesolvent is a good solvent for the gel polymer, porosity can only beintroduced into the gel by cross-linking or porogen. If, however, thereis present a solvent that is a thermodynamically poor solvent ornon-solvent, this solvent will act as a porogen. By combining solventsof different affinities to the gel polymer, from a good solvent througha poor solvent to a non-solvent, at different ratios, both porosity andpore dimensions can be altered. In general, the poorer the solvent orthe solvent mixture the higher the porosity and the sizes of macropores.Preferably, the solvent or solvent mixture for in situ polymerizationcontains poor solvent in the range from about 0% to about 100%, morepreferably from about 10% to about 90%. Examples of good solvents forpoly(2-acrylamido-2-methyl-1-propanesulfonic acid) are water andN,N-dimethylformamide. Examples of poor solvents include dioxane,hydrocarbons, esters, and ketones. An example of a good solvent forpoly(acrylamide) is water. Examples of poor solvents include dioxane,alcohols such as methanol, N,N-dimethylformamide, hydrocarbons, esters,and ketones. Preferably, the solvents used are miscible with water.

When the polymerization is carried out using a liquid vehicle thatcontains non-solvents or poor solvents, the resulting structure is oftenbuilt of clusters of agglomerated microspheres that form the body of themacroporous gel. The pores in such materials consist of the voidslocated between clusters (macropores), voids between microspheres in theclusters (mesopores), and pores inside the microspheres themselves(micropores).

Porogens can be broadly described as pore generating additives. Examplesof porogens that can be used in the gel-forming reaction includethermodynamically poor solvents or extractable polymers, for examplepoly(ethyleneglycol), or surfactants, or salts. Porogens are known inthe art, and a person skilled can determine, using standard experimentaltechniques and without exercise of any inventive faculty, which porogensare suitable to prepare macroporous gels for use in a desired membranes.

There is no simple way to predict accurately the structure parameters ofporous gels obtained under given conditions, but qualitative rules areavailable to give some guidance. Generally, the mechanism of porous gelformation via polymerization of one or more monomers and cross-linkersinvolves, as a first step, an agglomeration of polymer chains to givenuclei. The polymerization continues both in the nuclei and in theremaining solution to form microspheres which grow in size by capturingnewly precipitated nuclei and polymers from the solution. At some point,the microspheres become interconnected with each other in large clustersthat form the body of the macroporous gel. The poorer the solventquality the faster nucleation occurs during the gel-forming process. Ifthe number of nuclei formed is very large, as in the case of highconcentration of a polymerization initiator, smaller pores may beexpected. If, however, the number of nuclei is smaller and the reactionkinetics is such that the nuclei can grow larger, large pores are formedin the gel. High concentration of a cross-linker usually causes earlynucleation. The nuclei, however, may be too highly cross-linked to beable to swell with the monomers, grow and coalesce in clusters. This mayresult in very small pores. Because of the different ways that thepolymerization may proceed and the polymerization conditions may affectthe gel porous structure, a large variety of structures can be obtainedbut conditions for each of the structures need to be determinedexperimentally.

Composition of the Macroporous Gel-Filled Membranes

The macroporous gels can be formed through the in-situ reaction of oneor more polymerisable monomers with one or more cross-linkers, or of oneor more cross-linkable polymers with one or more cross-linker to form across-linked gel that has macropores of a suitable size. Suitablepolymerisable monomers include monomers containing vinyl or acrylgroups. For Donnan exclusion, there can be used vinyl or acryl monomerscontaining at least one polar and/or ionic functional group, orfunctional group that can be converted into ionic group. For biologicalaffinity there can be used vinyl or acryl monomers containing at leastone reactive functional group. Preferred polymerisable monomers includeacrylamide, 2-acryloxyethyltrimethylammonium chloride,N-acryloxysuccinimide, N-acryloyltris(hydroxymethyl)methylamine,2-aminoethyl methacrylate hydrochloride, N-(3-aminopropyl)methacrylamidehydrochloride, butyl acrylate and methacrylate, N,N-diethylacrylamide,N,N-dimethylacrylamide, 2-(N,N-dimethylamino)ethyl acrylate andmethacrylate, N-[3-(N,N-dimethylamino)propyl]methacrylamide,N,N-dimethylacrylamide, n-dodecyl acrylate, n-dodecyl methacrylate,dodecyl methacrylamide, ethyl methacrylate, 2-(2-ethoxyethoxy)ethylacrylate and methacrylate, 2,3-dihydroxypropyl acrylate andmethacrylate, glycidyl acrylate and methacrylate, n-heptyl acrylate andmethacrylate, 1-hexadecyl acrylate and methacrylate, 2-hydroxyethylacrylate and methacrylate, N-(2-hydroxypropyl)methacrylamide,hydroxypropyl acrylate and methacrylate, methacrylamide, methacrylicanhydride, methacryloxyethyltrimethylammonium chloride,2-(2-methoxy)ethyl acrylate and methacrylate, octadecyl acrylamide,octylacrylamide, octyl methacrylate, propyl acrylate and methacrylate,N-iso-propylacrylamide, stearyl acrylate, styrene, 4-vinylpyridine,vinylsulfonic acid, N-vinyl-2-pyrrodinone. Particularly preferredmonomers include dimethyldiallylammonium chloride,acrylamido-2-methyl-1-propanesulfonic acid (AMPS), (3-acrylamidopropyl)trimethylammonium chloride (APTAC), acrylamide, methacrylic acid (MAA),acrylic acid (AA), 4-styrenesulfonic acid and its salts, acrylamide,glycidyl methacrylate, diallylamine, and diallylammonium chloride.

The crosslinker may be, for example, a compound containing at least twovinyl or acryl groups. Examples of crosslinkers includebisacrylamidoacetic acid, 2,2-bis[4-(2-acryloxyethoxy)phenyl]propane,2,2-bis(4-methacryloxyphenyl)propane, butanediol diacrylate anddimethacrylate, 1,4-butanediol divinyl ether, 1,4-cyclohexanedioldiacrylate and dimethacrylate, 1,10-dodecanediol diacrylate anddimethacrylate, 1,4-diacryloylpiperazine, diallylphthalate,2,2-dimethylpropanediol diacrylate and dimethacrylate, dipentaerythritolpentaacrylate, dipropylene glycol diacrylate and dimethacrylate,N,N-dodecamethylenebisacrylamide, divinylbenzene, glyceroltrimethacrylate, glycerol tris(acryloxypropyl) ether,N,N′-hexamethylenebisacrylamide, N,N′-octamethylenebisacrylamide,1,5-pentanediol diacrylate and dimethacrylate, 1,3-phenylenediacrylate,poly(ethylene glycol) diacrylate and dimethacrylate, poly(propylene)diacrylate and dimethacrylate, triethylene glycol diacrylate anddimethacrylate, triethylene glycol divinyl ether, tripropylene glycoldiacrylate or dimethacrylate, diallyl diglycol carbonate, poly(ethyleneglycol) divinyl ether, N,N′-dimethacryloylpiperazine, divinyl glycol,ethylene glycol diacrylate, ethylene glycol dimethacrylate,N,N′-methylenebisacrylamide, 1,1,1-trimethylolethane trimethacrylate,1,1,1-trimethylolpropane triacrylate, 1,1,1-trimethylolpropanetrimethacrylate, vinyl acrylate, 1,6-hexanediol diacrylate anddimethacrylate, 1,3-butylene glycol diacrylate and dimethacrylate,alkoxylated cyclohexane dimethanol dicarylate, alkoxylated hexanedioldiacrylate, alkoxylated neopentyl glycol diacrylate, aromaticdimethacrylate, caprolacone modified neopentylglycol hydroxypivalatediacrylate, cyclohexane dimethanol diacrylate and dimethacrylate,ethoxylated bisphenol diacrylate and dimethacrylate, neopentyl glycoldiacrylate and dimethacrylate, ethoxylated trimethylolpropanetriarylate, propoxylated trimethylolpropane triacrylate, propoxylatedglyceryl triacrylate, pentaerythritol triacrylate, tris (2-hydroxyethyl)isocyanurate triacrylate, di-trimethylolpropane tetraacrylate,dipentaerythritol pentaacrylate, ethoxylated pentaerythritoltetraacrylate, pentaacrylate ester, pentaerythritol tetraacrylate, andcaprolactone modified dipentaerythritol hexaacrylate. Particularlypreferred cross-linking agents include N,N′,-methylenebisacrylamide,diethylene glycol diacrylate and dimethacrylate, trimethylolpropanetriacrylate, ethylene glycol diacrylate and dimethacrylate,tetra(ethylene glycol) diacrylate, 1,6-hexanediol diacrylate,divinylbenzene, poly(ethylene glycol) diacrylate.

The concentration of monomer in the macroporous gel can have an effecton the resiliency of the macroporous gel prepared. A low monomerconcentration can lead to a macroporous gel that is non-self supporting.Such non-self supporting gels might be advantageous as adsorbents, asthey could lead to gels having greater adsorption capacity. In someembodiments, the monomer concentration is 60% or less, for example about60, 50, 40, 30, 20, 10 or 5%.

When a cross-linkable polymer is used, it can be dissolved and reactedin-situ in the support with a cross-linking agent to form themacroporous gel. Suitable cross-linkable polymers includepoly(ethyleneimine), poly(4-vinylpyridine), poly(vinylbenzyl chloride),poly(diallylammonium chloride), poly(glycidyl methacrylate),poly(allylamine), copolymers of vinylpyridine anddimethyldiallylammonium chloride, copolymers of vinylpyridine,dimethyladiallylammonium chloride, or(3-acrylamidopropyl)trimethylammonium chloride with glycidyl acrylate ormethacrylate, of which poly(ethyleneimine), poly(diallylammoniumchloride), and poly(glycidyl methacrylate) are preferred. Use ofcross-linkable polymers instead of monomers can, in some instances,require a decrease in the concentration of cross-linking agent. In orderto retain the large size of the pores in the gel with a lowercross-linking agent concentration, a porogen can be added to the mixtureused to prepare the macroporous gel.

The cross-linking agent for reaction with the cross-linkable polymer isselected from molecules containing two or more reactive groups that canreact with an atom or group of atoms in the polymer to be cross-linked,such as epoxy groups or alkyl/aryl halides that can react with nitrogenatoms of polyamines, or amine groups that can react with alkyl/arylhalides or epoxy groups of glycidyl-group-containing polymers to be insitu cross-linked. Suitable cross-linkers include ethylene glycoldiglycidyl ether, poly(propylene glycol) diglycidyl ether,1,3-dibromopropane, 1,4-dibromobutane, 1,5-dibromopentane,1,6-dibromohexane, α,α′-dibromo-p-xylene, α,α′-dichloro-p-xylene,1,4-dibromo-2-butene, piperazine, 1,4-diazabicyclo[2.2.2]octane,1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane,1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane,1,8-diaminooctane.

It is also possible to modify polymers containing reactive groups suchas an amino, hydroxyl, carboxylic acid, carboxylic acid ester, or epoxygroups with reagents to introduce vinyl groups that can be subsequentlypolymerized by treatment with a polymerization initiator to form amacroporous gel. Examples of suitable vinyl groups that can beintroduced include vinylbenzene derivatives, allyl derivatives, acrolyland methacrolyl derivatives. The cross-linking of these vinylsubstituted polymers can in some instances be facilitated by theintroduction of further monomers such as acrylamide, N-vinylpyrrolidone,acrylic and methacrylic acids and their salts.

Macromonomers can also be used as monomers or as cross-linking agents.Macromonomers can be polymers or oligomers that have one(monofunctional) or more (cross-linking agent) reactive groups, often atthe ends, which enable them to act as a monomer or a cross-linker. Formonomers, each macromonomer molecule is attached to the main chain ofthe final polymer by reaction of only one monomeric unit in themacromonomer molecule. Examples of macromonomers include poly(ethyleneglycol)acrylate and poly(ethylene glycol)methacrylate, while examples ofpolyfunctional macromonomers include poly(ethylene glycol)diacrylate andpoly(ethylene glycol)dimethacrylate. Macromonomers preferably havemolecular weights of about 200 Da or greater.

Many macroporous gels can be prepared, including neutral hydrogels,charged hydrogels, polyelectrolyte gels, hydrophobic gels, and neutraland functional gels.

If the gel selected is a neutral hydrogel or a charged hydrogel forwhich water is the swelling liquid medium, the resulting supportedmacroporous gel is normally quite hydrophilic. Hydrophilic macroporousgel-filled membranes are preferred as they provide better flowcharacteristics and impart anti-fouling tendencies to the membranes.Examples of suitable hydrogels include cross-linked gels of poly(vinylalcohol), poly(acrylamide), poly(isopropylacrylamide),poly(vinylpyrrolidone), poly(hydroxymethyl acrylate), poly(ethyleneoxide), copolymers of acrylic acid or methacrylic acid with acrylamide,isopropylacrylamide, or vinylpyrrolidone, copolymers ofacrylamide-2-methyl-1-propanesulfonic acid with acrylamide,isopropylacrylamide, or vinylpyrrolidone, copolymers of(3-acrylamidopropyl) trimethylammonium chloride with acrylamide,isopropylacrylamide, or N-vinylpyrrolidone, copolymers ofdiallyldimethylammonium chloride with acrylamide, isopropylacrylamide,or vinylpyrrolidone. Preferred hydrogels include cross-linked poly(vinylalcohol), poly(acrylamide), poly(isopropylacrylamide) andpol(vinylpyrrolidone) and cross-linked copolymers of neutral monomerssuch as acrylamide or N-vinylpyrrolidone with charged monomers such asacrylamide-2-methyl-1-propanesulfonic acid or diallyldimethylammoniumchloride.

The macroporous gels can be selected to comprise polyelectrolytes. Likethe charged hydrogels, polyelectrolyte gels give hydrophilic macroporousgel-filled membranes, and they also carry a charge. The polyelectrolytegel can be selected, for example, from cross-linkedpoly(acrylamido-2-methyl-1-propanesulfonic acid) and its salts,poly(acrylic acid) and its salts, poly(methacrylic acid) and its salts,poly(styrenesulfonic acid) and its salts, poly(vinylsulfonic acid) andits salts, poly(alginic acid) and its salts,poly[(3-acrylamidopropyl)trimethylammonium] salts,poly(diallyldimethylammonium) salts, poly(4-vinyl-N-methylpyridinium)salts, poly(vinylbenzyl-N-trimethylammonium) salts, poly(ethyleneimine)and its salts. Preferred charged gels include cross-linked poly(acrylicacid) and its salts, poly(methacrylic acid) and its salts,poly(acrylamido-2-methyl-1-propanesulfonic acid) and its salts,poly[(3-acrylamidopropyl)trimethylammonium] salts,poly(diallyldimethylammonium) salts, and poly(4-vinylpyridinium) salts.

One of the differences between charged gels and polyelectrolyte gels isthat the repeating monomer in the polyelectrolyte gel bears a charge,while in the charged gel, the charge is found in a co-polymerized unitthat is randomly distributed through the polymer. The monomer used toform the polyelectrolyte gel or the co-polymer in the charged gel thatbears a charge usually has a charge bearing group, but it can also be anon-charge-bearing group that can become charged in a post-gelationprocess (e.g. quaternization of nitrogen bearing groups). Examples ofpolymers that can become charged include poly(4-vinylpyridine) which canbe quaternized with various alkyl and alkylaryl halides. Suitable alkylhalides include those having up to 8 carbon atoms, for example methyliodide, ethyl bromide, butyl bromide, and propyl bromide. Suitablealkylaryl halides include benzyl halides, especially benzyl chloride andbenzyl bromide. Another polymer that can become charged ispoly(vinylbenzyl chloride), which can be quaternized with variousamines, for example, lower alkylamines or aromatic amines such astriethylamine, pyridine, azabicyclo[2.2.2]octane, N-methylpyrrolidine,and N-methylpiperidine, and lower hydroxyalkylamines, for exampletriethanolamine. Yet another polymer that can become charged ispoly(glycidyl methacrylate) or poly(glycidyl acrylate), which can reactwith various amines, for example lower alkylamines such as diethylamineand triethylamine, azabicyclo[2.2.2]octane, N-methylpyrrolidine, andN-methylpiperidine. Alternatively, glycidyl moieties can be converted tosulfonic acid groups by reaction with, for example alkali metal sulfitessuch as sodium sulfite. A person skilled in the art will appreciate thatthere are other polymers that are, or can be rendered, charge-bearing.

The macroporous gel can be selected to comprise hydrophobic monomers topermit separations in organic solvents, for example hydrocarbons,especially liquid paraffins such as hexanes. Hydrophobic monomers, suchas styrene and its derivatives, for example an alkyl substituted styrenederivative such as para-tertbutyl styrene, can be used to preparehydrophobic macroporous gels. Copolymers of these monomers can be used.

A macroporous gel comprising hydrophobic monomers can be used to capturemolecules from fluids passing through the pores by hydrophobicinteractions.

As stated above, the macroporous gels can also be selected to comprisereactive functional groups that can be used to attach ligands or otherspecific binding sites. These functional macroporous gels can beprepared from cross-linked polymers bearing functional groups, forexample epoxy, anhydride, azide, reactive halogen, or acid chloridegroups, that can be used to attach the ligands or other specific bindingsites. Examples include cross-linked poly(glycidyl methacrylate),poly(acrylamidoxime), poly(acrylic anhydride), poly(azelaic anhydride),poly(maleic anhydride), poly(hydrazide), poly(acryloyl chloride),poly(2-bromoethyl methacrylate), poly(vinyl methyl ketone).Functionality that can be introduced can take the form of antibodies orfragments of antibodies, or alternatively, chemical mimics such as dyes.Functional gels are attractive in biomolecule purifications orseparations, as they can offer preferential binding to certain moleculesby binding to active sites, while being non-reactive to other molecules,even when there is no significant difference in size between themolecules, examples being affinity ligands selected to bind with someproteins but not others. Affinity ligands that can be attached to porousgels via reactive groups include amino acid ligands such asL-phenylalanine, tryptophan, or L-histidine to separate γ-globulins andimmunoglobulins, antigen and antibody ligands such as monoclonalantibodies, protein A, recombinant protein A, protein G, or recombinantprotein G to separate immunoglobulins from different media, dye ligandssuch as cibaron blue or active red to separate albumins and variousenzymes, metal affinity ligands such as complexes of iminodiacetic acid(IDA) ligand with Cu²⁺, Ni²⁺, Zn²⁺, or Co²⁺ to separate various proteinssuch as histidine, lysozyme, or tryptophan from various media.

Responsive Macroporous Gels

Polymers that change their conformation in response to changes inenvironmental conditions are known. By incorporating the properties ofsuch polymers in the macroporous gel, a macroporous gel-filled membranewith dynamic pore-size is obtained. These membranes having responsivecharacteristics are substantially the same as the macroporous gel-filledmembranes described above, except that at least one of the monomers orpolymers that form the macroporous gel has a chemical structure whichfacilitates changes in pore-size.

The changes in the pore-size of the macroporous gel are due to thephysical relationship between the support member and the macroporousgel. The macroporous gel-filled membrane can be described as havingthree distinct zones: (a) the support member, which ideally does notchange shape, (b) the incorporated macroporous gel that “fills” thepores of the support member, and (c) the volume within the macropores ofthe gel, which volume is filled with water or a solvent and in which isfound very little or no gel polymer. Under pressure, hydraulic flowoccurs through the macropores of the gel, and the flux through themembrane is related to the number of pores in the macroporous gel, theradius of these pores, and the tortuosity of the path of the pores inthe macroporous gel through the macroporous gel-filled membrane.

As the degree of swelling of the macroporous gel is changed by anenvironmental stimulus, the total volume occupied by the macroporous gelis constrained by the fixed total volume defined by the support member.As the overall volume of the macroporous gel is constrained by thesupport member, by necessity the volume fraction of the gel expands intothe area defined by macropores in the gel. As the number of macroporesand their tortuosity remain essentially constant with the change involume fraction of the macroporous gel, the diameter or radius of themacropores themselves must change. If the macroporous or structured gelwere unconfined, the environmentally induced changes would cause thetotal volume of the swollen gel to change. As such it would not followin this unconfined case that the changes would result in a controllablechange in pore-size of the macroporous gel.

The reason behind the change in volume of the macroporous gel is relatedto interactions between the polymer structures that form the gels, orthe interactions between the polymer chains and the solvents or solutespresent in the solvent that diffuse into the gel. The changes in thevolume occupied by the gel are linked to the conformation adopted by thepolymer chains that form the macroporous gels. The natural tendency ofthe polymer chains is to coil around themselves, which leads to a gelhaving a smaller volume. If the polymer chains within the gel can bemanipulated to uncoil and form a more rigid backbone, the overall volumeof the gel will increase. It is thus this coiling/uncoiling which isaffected by the environmental stimuli that are applied to the responsivemacroporous gel-filled membrane.

The volume changes of the pores can either be “continuous” or“discontinuous”. A continuous volume change takes place over arelatively large change in the triggering environmental condition andwhere there exists at least one stable volume near the transitionbetween the swollen and collapsed state. Preferably, a continuous volumechange will go through a multitude of stable transition volumes betweenthe swollen and the collapsed state. A discontinuous volume change ingels is characterised by the reversible transition from swollen tocollapsed state taking place over an extremely small change in thetriggering environmental condition, for example, less than 0.1 pH unitor 0.1 degree Celsius. Gels exhibiting discontinuous volume change arecalled “phase-transition” gels and systems or devices with such gels areoften called “chemical valves”. Preferably, the responsive macroporousgels according to this embodiment of the invention undergo a“continuous” volume change through discrete stable volumes that can beutilized to control the pore-size of the gel.

Of the environmental stimuli that can be used to change the pore-size inthe responsive macroporous, mention is made of pH, specific ions, ionicstrength, temperature, light, electric fields, and magnetic fields. Theeffect of each stimulus, and examples of monomers that react to such astimulus, will be described in more detail below.

One stimulus that can be utilised to change the pore-size of responsivemacroporous gel is the pH of the solution being passed through the poresof the gel. A change in the pH of the solution will affect the pore-sizeof the gel if the gel comprises weak acids or weak bases. In such cases,the natural tendency of the polymer chain within the gel to coil arounditself will be balanced by the repulsion between the charged groups(weak acidic or basic groups) along the length of the polymer chain.Variations in the amount of charge along the chain cause large changesin conformation of the polymer chain, which in turn causes changes inthe volume occupied by the gel. Changes in the pH of the solution areeffective at controlling the amount of repulsion along the polymerchain, as they change the degree of ionisation of the charged groups. Agel comprising weak acid groups becomes less ionised as the pH islowered and the gel contracts. Conversely, a weak base becomes moreionised as the pH is lowered and the chain elongates or stretches togive a swollen gel.

Examples of monomers that have weak acid functionality include acrylicacid, methacrylic acid, itaconic acid, 4-vinylbenzoic acid,bisacrylamidoacetic acid, and bis(2-methacryloxyethyl) phosphate.Examples of monomers that have weak base functionality include2-aminoethyl methacrylate hydrochloride, N-(3-aminopropyl)methacrylamidehydrochloride, 2-(tert-butylamino)ethyl methacrylate, diallylamine,2-(N,N-diethylamino)ethyl methacrylate, 2-(N,N-dimethylamino)ethylacrylate, 2-(N,N-dimethylamino)ethyl methacrylate, 1-vinylimidazole, and4-vinylpyridine. Glycidyl methacrylate derivatizedhyaluronate-hydroxyethyl acrylate based hydrogels can also be used toprepare macroporous gel-filled membranes that are pH responsive [InukaiM., Jin Y., Yomota C., Yonese M., Chem. Pharm. Bull., (2000),48:850-854; which is hereby incorporated by reference].

Variations in pH have little effect on the degree of ionisation ofstrong acids and bases, and as such, only drastic variations in pH caneffect pore-size changes in gels comprising these functionalities.

Another stimulus that can be utilised for changing the pore-size of aresponsive macroporous gel is the salt concentration of the solutionbeing passed through the pores of the gel. Similarly to variations inpH, variations in salt concentration will effect pore-size variations inmacroporous gels that comprise weak acidic or weak basic groups. Thereason for the changes in pore-size, however, does differ slightly. Theaddition of an ionic solute has the ability to shield the charged groupsfound on the polymer chain in the gel by the formation of ion-pairs.This lessens the coulombic repulsion between the adjacent chargedgroups, which allows the chain to relax into a coiled conformation. Anincrease in salt concentration will shield both a weak acid group and aweak base group. Therefore, when the salt concentration is increased,for example by adding a concentrated salt solution to the bulk solutionbeing passed through the macroporous gel-filled membrane, the shieldingeffect of the additional ions leads to an increase in pore size.Alternatively, a decrease in salt concentration, such as obtained bydiluting the bulk solution being passed through the macroporousgel-filled membrane, will lead to less shielding and a smaller poresize.

Changes in salt concentration can also be used with macroporous gelsthat comprise strong acid groups and strong basic groups, as thesegroups are also shielded by the presence of free ionic species.

Examples of monomers that bear weak acid or base groups are listedabove. Examples of monomers that have strong acid functionality include2-acrylamido-2-methyl-1-propanesulfonic acid, sodium2-methyl-2-propene-1-sulfonate, sodium styrenesulfonate, and sodiumvinylsulfonate. Examples of monomers that have strong basicfunctionality include 2-acryloxyethyltrimethylammonium chloride,diallyldimethylammonium chloride, methacrylamidopropyltrimethylammoniumchloride, and 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride.

Ionic functionality (weak/strong acids and bases) may also be introducedin macroporous gels that do not originally bear charged functionalitiesbut that bear instead reactive groups that can be converted into ionicor ionisable moieties in a post-polymerization treatment. Suitablemonomers with reactive groups include acrylic anhydride, allyl glycidylether, bromostyrene, chloromethylstyrene, chlorostyrenes, glycidylacrylate, glycidyl methacrylate, 4-hydroxybutyl methacrylate,2-hydroxyethyl acrylate, methacryloyl chloride. For example, amacroporous gel comprising a glycidyl acrylate or methacrylate group canbe treated with diethylamine to introduce weak base functionality orwith sodium sulfite in an iso-propanol/water mixture to introduce strongacid (sulfonic acid) functionality.

Another stimulus that can be used to change the pore-size of aresponsive macroporous gel is the temperature of the gel. Variousmethods are available for changing the temperature of the macroporousgel, one of which includes changing the temperature of a liquid flowingthrough the pores of the macroporous gel. While the change in overallgel volume for temperature dependent gels is again due to the control ofthe coiling or uncoiling of the polymer chains that form the gel, thecontraction or expansion of the gel is not linked to the presence ofcharged groups on the polymer chain. For temperature dependant gels, theamount solvation of the polymer chain controls the conformation of thepolymer chain. At lower temperatures the chains are solvated, whichallows an elongated conformation of the polymer chain. As thetemperature is increased, an entropic desolvation takes place causingthe chains to coil and contract. Therefore, increases in temperaturelead to larger pore sizes in the gel while decreases in temperature leadto smaller pore sizes.

Macroporous gels that comprise hydrophobic monomers are most suitablefor use in temperature dependant systems, as salvation effects aremarkedly observed for polymers that have hydrophobic functionality.Examples of monomers that have hydrophobic functionality includeN-(propyl)acrylamide, N-(tert-butyl)acrylamide, butyl acrylates, decylacrylates, decyl methacrylates, 2-ethylbutyl methacrylate, n-heptylacrylate, n-heptyl methacrylate, 1-hexadecyl acrylate, 1-hexadecylmethacrylate, n-hexyl acrylate, n-hexyl methacrylate, andN-(n-octadecyl)acrylamide. Gels displaying thermal response can also beprepared from sulphated hyaluronic acid-based gels (see Barbucci R.,Rappuoli R., Borzacchiello A., Ambrosio L., J. Biomater. Sci.-Polym.Ed., (2000), 11:383-399), incorporated herein by reference.

Light is another stimulus that can be used to change the pore-size ofthe responsive macroporous gel. Light induced changes are due tophotoisomerizations in the backbone or the side-chains of the polymerchains that form the gel. These photoisomerizations cause a change ineither the conformation and local dipole moment, or in the degree ofionisation through light induced electron transfer reactions. One typeof monomer that is suitable for use in light controlled systemscomprises unsaturated functionalities than can undergo a trans-cisisomerization on irradiation. Examples of photoresponsive monomers thatgo through cis-trans conformation and dipole changes include4-(4-oxy-4′-cyanoazobenzene)but-1-yl methacrylate,6-(4-oxy-4′-cyanoazobenzene)hex-1-yl methacrylate,8-(4-oxy-4′-cyanoazobenzene)oct-1-yl methacrylate,4-[ω-methacryloyloxyoligo(ethyleneglycol)]-4′-cyanoazobenzene,4-methacryloyloxy-4′-{2-cyano-3-oxy-3-[ω-methoxyoligo(ethyleneglycol)]prop-1-en-1-yl}azobenzene,and methacrylate monomers containing a mesogenic group and aphotochromic para-nitroazobenzene group. It is also possible toincorporate the photoresponsive moeity in the crosslinker instead of themonomer. Examples of photoresponsive crosslinkers that go throughcis-trans conformation and dipole changes include4,4′-Divinylazobenzene, N,N′-bis(β-styrylsulfonyl)-4,4′-diaminoazobenzene,4,4′-bis(methacryloylamino)azobenzene, 4,4′-dimethacryloylazobenzene,and bis((methacryloyloxy)methyl)spirobenzopyran.

The pore-size of the gel can also be altered by subjecting themacroporous gel to an electric field or to an electrical current. Theresponse of the gel to electrochemical current changes is closelyrelated to the pH systems described above. This close relationship isdue to the fact that the passage of an electrochemical current throughan aqueous system causes a “water splitting” reaction, which reactionleads to changes in the pH of the aqueous system. Electrical current canbe passed through a macroporous gel-filled membrane e.g. by placing anelectrode at either end of the macroporous gel-filled membrane. Whencurrent differential is applied to the electrodes, water molecules willseparate and concentrations of H⁺ and HO⁻ will increase at theirrespective electrodes. As described earlier, changes in pH can be usedto control the pore-size of macroporous gels that comprise weak acid orweak base functionalities, which control is linked to the relationshipbetween the ionisation of these functionalities and thecoiling/uncoiling of the polymer chains that form the gel. Examples ofweak acidic and weak basic monomers are given above.

Changes in gel volume due to fluctuations in an electrical field havebeen previously observed, such as in Murdan S., J. Control. Release,(2003), 92:1-17; and in Jensen M., Hansen P. B., Murdan S., Frokjaer S.,Florence A. T., Eur. J. Pharm. Sci., (2002), 15:139-148, which arehereby incorporated by reference. While the exact process through whichthe gel volume is changed by the application of an electrical field isnot yet well defined, the volume change itself is well documented.Chondroitin 4-sulphate (CS) is an example of a monomer that isresponsive to electrical field fluctuations.

In some embodiments, the various stimuli response systems can becombined to offer gels that respond to more than one stimulus. Anexample of such a combined system can be prepared by combining a chargedpolymer (weak/strong acid or base) with a hydrophobic monomer. Themacroporous gel resulting from such a combination will display responsesto changes in salt concentration, changes in solution pH (when weakacids or bases are used), and changes in temperature. When combiningdifferent monomers, it is possible that the responsiveness of the gel toa single of the stimuli will be diminished, as the concentration of themonomer that responds to that particular stimulus will be lowered in thegel.

The magnitude of the response expressed by the macroporous gels, whenvarious stimuli are applied to the gel, depends many differentvariables, a few of which are discussed below:

The responsiveness of the macroporous gel is dependent on theconcentration of the crosslinking agent. Generally, as the concentrationof cross-linking agent is increased, the size of the macropores in theresponsive gel is also increased, but the range of pore-size changes isdecreased. This relationship is fairly straightforward, as a higherconcentration of crosslinks within the gel will limit the amount ofcoiling and uncoiling that will be available to the responsive gel. Themolar ratio of crosslinking agent(s) to monomer(s) may be in the rangeof from about 5:95 to about 40:60, preferably in the range of from about7.5:92.5 to about 10:90, and more preferably in the range of from about10:90 to about 25:75.

Certain stimuli naturally evoke a broader range of response in the gel,as they more effectively affect the conformation of the polymer chainsthat form the gel. For example, variations in pH or temperature evoke astrong response from the appropriate macroporous gels, while changes insalt concentrations and light intensity evoke a slightly smallerresponse.

The concentration of the responsive monomer in the gel also affects thelevel of response demonstrated by the gel. Preferably, the responsivemacroporous gels are composed of one or more responsive monomers and ofone or more neutral monomers. The presence of a neutral monomer isimportant in those systems that have a very strong response to changesin the environmental conditions, as such systems often displaydiscontinuous responses in pore-size (valve-effects). Addition of aneutral monomer attenuates the response, permitting a more controlledchange in pore-size. Preferably, the molar ratio of the neutral monomersto the molar ratio of responsive monomers in the responsive macroporousgel is in the range from 5:95 to 95:5, more preferably in the range from25:75 to 75:25, and more preferably in the range from 40:60 to 60:40.Suitable neutral monomers include acrylamide, N-acryloylmorpholine,N-acryloxysuccinimide, 2-acrylamido-2-(hydroxymethyl)-1,3-propanediol,N,N-diethylacrylamide, N, N-dimethylacrylamide, 2-(2-ethoxyethoxy)ethylacrylate, 2-ethoxyethyl methacrylate, 2,3-dihydroxypropyl methacrylate,2-hydroxyethyl methacrylate, N-(2-hydroxypropyl)methacrylamide,hydroxypropyl methacrylate, methacrylamide, N-[tris(hydroxymethyl)methyl]-1-methacrylamide, N-methylmethacrylamide,N-methyl-N-vinylacetamide, poly(ethylene glycol) monomethacrylate,N-iso-propylacrylamide, N-vinyl-2-pyrrolidone.

Porous Support Member

A variety of materials can be used to form the support member; however,apart from materials such as cellulose and some of its derivatives, mostof these materials are strongly or relatively hydrophobic. Hydrophobicfiltration membranes are not usually desired for use with aqueoussystems, as they can lead to higher membrane fouling tendencies. Themore inert and cheaper polymers such as polyolefins, for example(poly(ethylene), poly(propylene) poly(vinylidene difluoride)) can beused to make microporous membranes, but these materials are veryhydrophobic. In some embodiments, the hydrophobicity of the supportmember does not affect the degree of fouling experienced by themacroporous gel-filled membrane as the flow of liquid through themacroporous gel-filled membrane takes place primarily in the macroporesof the gel.

In some embodiments, the porous support member is made of polymericmaterial and contains pores of average size between about 0.1 and about25 μm, and a volume porosity between 40 and 90%. Many porous substratesor membranes can be used as the support member but the support ispreferably a polymeric material, and it is more preferably a polyolefin,which, while hydrophobic, is available at low cost. Extended polyolefinmembranes made by thermally induced phase separation (TIPS), ornon-solvent induced phase separation are mentioned. Hydrophilic supportscan also be used, including natural polymers such as cellulose and itsderivatives. Examples of suitable supports include SUPOR®polyethersulfone membranes manufactured by Pall Corporation,Cole-Parmer® Teflon® membranes, Cole-Parmer® nylon membranes, celluloseester membranes manufactured by Gelman Sciences, Whatman® filter andpapers.

In some other embodiments the porous support is composed of woven ornon-woven fibrous material, for example a polyolefin such aspolypropylene. An example of a polypropylene non-woven material iscommercially available as TR2611A from Hollingsworth and Vose Company.Such fibrous woven or non-woven support members can have pore sizeslarger than the TIPS support members, in some instances up to about 75μm. The larger pores in the support member permit formation ofmacroporous gel-filled membranes having larger macropores in themacroporous gel. Macroporous gel-filled membrane with larger macroporescan be used, for example, as supports on which cell growth can becarried out. Non-polymeric support members can also be used, such asceramic-based supports. The porous support member can take variousshapes and sizes.

In some embodiments, the support member is in the form of a membranethat has a thickness of from about 10 to about 2000 μm, more preferablyfrom 10 to 1000 μm, and most preferably from 10 to 500 μm. In otherembodiments, multiple porous support units can be combined, for example,by stacking. In one embodiment, a stack of porous support membranes, forexample from 2 to 10 membranes, can be assembled before the macroporousgel is formed within the void of the porous support. In anotherembodiment, single support member units are used to form macroporousgel-filled membrane, which are then stacked before use.

Preparation of Macroporous Gel-Filled Membranes

The macroporous gel-filled membranes can be prepared by simple, singlestep methods. These methods can, in some instances, use water or otherbenign solvents, such as methanol, as the reaction solvent. The methodsalso have the benefit of using rapid processes that lead to easier andcontinuous manufacturing possibilities. The macroporous gel-filledmembrane is also potentially cheap.

The macroporous gel-filled membranes can be prepared, for example, bymixing one or more monomers, one or more polymers, or mixtures thereof,one or more cross-linking agents, optionally one or more initiators andoptionally one or more porogens, in one or more suitable solvents. Thesolution produced is preferably homogeneous, but a slightlyheterogeneous solution can be used. The mixture is then introduced intoa suitable porous support, where a gel forming reaction takes place.Suitable solvents for the gel forming reaction include, for example,water, dioxane, dimethylsulfoxide (DMSO), dimethylformamide (DMF),acetone, ethanol, N-methylpyrrolidone (NMP), tetrahydrofuran (THF),ethyl acetate, acetonitrile, toluene, xylenes, hexane,N-methylacetamide, propanol, and methanol. It is preferable to usesolvents that have a higher boiling point, as these solvents reduceflammability and facilitate manufacture. It is also preferable that thesolvents have a low toxicity, and they can be readily disposed of afteruse. An example of such a solvent is dipropyleneglycol monomethyl ether(DPM).

In some embodiments, it is possible to use dibasic esters (esters of amixture of dibasic acids) as the solvent. Dibasic esters (DBEs) areespecially suitable for preparing gels based on polyacrylamide monomers.This solvent system has an unexpected characteristic in that it ispoorly soluble in water, which differs from the other solvents usedwhich are essentially completely water miscible. While water misciblesolvents offer advantages in terms of solvent removal after fabrication,water immiscible solvents such as DBE's are good replacements, incertain cases, for solvents such as dioxane that are volatile,flammable, and toxic.

In some embodiments, components of the gel forming reaction reactspontaneously at room temperature to form the macroporous gel. In otherembodiments, the gel forming reaction must be initiated. The gel formingreaction can be initiated by any known method, for example throughthermal activation or U.V. irradiation. The reaction is more preferablyinitiated by U.V. irradiation in the presence of a photoinitiator, asthis method has been found to produce larger macropores in the gel, andit accelerates the gel forming reaction more than the thermal activationmethod. Many suitable photoinitiators can be used, of which2-hydroxy-1[4-2(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure2959*), and 2,2-dimethoxy-2-phenylacetophenone (DMPA) are preferred.Other suitable photoinitiators include benzophenone, benzoin and benzoinethers such as benzoin ethyl ether and benzoin methyl ether,dialkoxyacetophenones, hydroxyalkylphenones, α-hydroxymethyl benzoinsulfonic esters. Thermal activation requires the addition of a thermalinitiator. Suitable thermal initiators include1,1′-azobis(cyclohexanecarbonitrile) (VAZO® catalyst 88),azobis(isobutyronitrile) (AIBN), potassium persulfate, ammoniumpersulfate, and benzoyl peroxide.

If the reaction is to be initiated by U.V. irradiation, a photoinititoris added to the reactants of the gel forming reaction, and the supportmember containing the mixture of monomer, cross-linking agent andphotoinitiator is subjected to U.V. irradiation at wavelengths of from250 nm to 400 nm, for a period of a few seconds to a few hours. Withcertain photoinitiators, visible wavelength light may be used toinititate the polymerization. To permit the initiation, the supportmaterial must have a low absorbance at the wavelength used, to permittransmittance of the UV rays through the support. Preferably, thesupport and macroporous gel reagents are irradiated at 350 nm for a fewseconds to up to 2 hours.

Preferably, thermally initiated polymerization is carried out at 60-80°C. for a few minutes up to 16 hours.

The rate at which polymerization is carried out has an effect on thesize of the macropores obtained in the macroporous gel. As discussedearlier, when the concentration of cross-linker in a gel is increased tosufficient concentration, the constituents of the gel begin to aggregateto produce regions of high polymer density and regions with little or nopolymer, which latter regions are referred to as “macropores” in thepresent specification. It is this mechanism which is affected by therate of polymerization. When polymerization is carried out slowly, suchas when a low light intensity in the photopolymerization, theaggregation of the gel constituents has more time to take place, whichleads to larger pores in the gel. Alternatively, when the polymerizationis carried out at a high rate, such as when a high intensity lightsource is used, there is less time available for aggregation and smallerpores are produced.

Once the macroporous gel-filled membranes are prepared, they can bewashed with various solvents to remove any unreacted components and anypolymer or oligomers that are not anchored within the support. Solventssuitable for the washing of the macroporous gel-filled membrane includewater, acetone, methanol, ethanol, and DMF.

It must be noted that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise. Unless defined otherwiseall technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs.

EXAMPLES

The following examples are provided to illustrate the invention. It willbe understood, however, that the specific details given in each examplehave been selected for purpose of illustration and are not to beconstrued as limiting the scope of the invention. Generally, theexperiments were conducted under similar conditions unless noted.

Experimental Materials Used

The monomers used were acrylamide (AAM),2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS),(3-acrylamidopropane)trimethylammonium chloride (APTAC),diallyldimethylammonium chloride (DADMAC), ethylene glycoldimethacrylatecrylate (EDMA), glycidyl methacrylate (GMA),N,N′-methylenebisacrylamide (BIS), methacrylic acid (MAA), acrylic acid(AA), and trimethylolpropane triacrylate (TRIM). The polymers used werebranched poly(ethylene imine) (BPEI) of an average molecular weight (MW)of 25000 Da, poly(ethylene glycol) (PEG) of average molecular weight of200, 1000, 2000, 4000 and 10000 Da, and poly(allylammoniumhydrochloride) (PAH) of an average molecular weight of 60000 Da. Thecross-linker used for BPEI was ethylene glycol diglycidyl ether (EDGE).

The solvents used were di(propylene glycol) methyl ether, 97%, mixtureof isomers (DPM) (Aldrich), cyclohexanol (CHX), methylene chloride(CH₂Cl₂), deionized water, 1,4-dioxane, N,N-dimethylformamide (DMF),dodecanol (DDC), glycerol, methanol, 1-octanol, and 1-propanol.

The free radical polymerization initiators used were2-hydroxy-1-[4-(2-hydoxyethoxy)phenyl]2-hydroxy-2-methyl-1-propane-1-one(Irgacure® 2959), 2,2-dimethoxy-2-phenylacetophenone (DMPA), and1,1′-azobis(cyclohexanecarbonitrile) (VAZO® catalyst 88).

The mesh-spacers used were Naltex (Austin, Tex., USA) symmetricalfiltration netting made of polypropylene of thickness of 500 μm and meshopening of 1000 μm and a polyester mesh (Polyester Monofilament) ofthickness of 350 μm and mesh opening of 500 μm obtained from Great LakesFilters Inc. Hillsdale Mich.

Proteins used were bovine serum albumin (BSA), lysozyme, human serumalbumin (HSA) and human immunoglobulin (HIgG).

Other chemicals used were acryloyl chloride, hydrochloric acid, sodiumazide, sodium chloride, sodium hydroxide, triethylamine,tris(hydroxymethyl)aminomethane (TRIS), 4-morpholineethanesulfonic acid(MES) and buffers (Tris Buffer).

The porous supports used were poly(propylene) thermally induced phaseseparation (TIPS) membranes PP1545-4 with an average pore diameter of0.45 μm, thickness of 125 μm, and porosity of 85 vol-%, produced by 3MCompany, and PP1183-3X of an average pore diameter of 0.9 μm, thicknessof 87 μm, and porosity of 84 vol-%, both produced by 3M Company, andnon-woven meltblown poly(propylene) TR2611A of a mean pore flow diameterof 6.5 μm, thickness of 250 μm, and porosity of 89.5 vol-% produced byHollingworth & Vose Company.

Preparation of Macroporous Gel-Filled Membranes

The macroporous gel-filled membranes can be prepared according to thefollowing general procedure. A weighed support member was placed on apoly(ethylene terephthalate) (PET) or poly(ethylene) (PE) sheet and amonomer or polymer solution was applied the sample. The sample wassubsequently covered with another PET or PE sheet and a rubber rollerwas run over the sandwich to remove excess solution. In situ gelformation in the sample was induced by polymerization initiated byirradiation with the wavelength of 350 nm for the period of 10 to 120minutes or by heating the sandwich at 60-80° C. for 2 hours. Theirradiation was typically carried out using a system containing four 12″long lamps, approx. 1.5″ spaced and emitting light at 365 nm with theoutput energy of approx. 0.1 Watt/inch. The system was equipped with asmall fan to dissipate the heat (no other temperature control). Theirradiated sample was located at approx. 5″ distance from the lamps. Incase when preformed polymer and in situ cross-linking was used to formthe gel, the sandwich was left at room temperature until thecross-linking reaction was completed, typically for 2-16 hours. Theresulting macroporous gel-filled membrane was thoroughly washed with asuitable solvent or a sequence of solvents and stored in a 0.1 wt-%aqueous solution of sodium azide to prevent bacterial growth. In orderto determine the amount of gel formed in the support, the sample wasdried in vacuum at room temperature to a constant mass. The mass gaindue to gel incorporation was calculated as a ratio of an add on mass ofthe dry gel to the initial mass of the porous support.

Flux Measurements

Water flux measurements through the macroporous gel-filled membraneswere carried out after the samples had been washed with water. As astandard procedure, a sample in the form of a disk of diameter 7.8 cmwas mounted on a sintered grid of 3-5 mm thickness and assembled into acell supplied with compressed nitrogen at a controlled pressure. Thecell was filled with deionized water or another feed solution and adesired pressure was applied. The water that passed through themacroporous gel-filled membrane in a specified time was collected in apre-weighed container and weighed. All experiments were carried out atroom temperature and at atmospheric pressure at the permeate outlet.Each measurement was repeated three or more times to achieve areproducibility of ±5%.

The water flux, Q_(H20) (kg/m²h), was calculated from the followingrelationship:

$Q_{H_{2}O} = \frac{\left( {m_{1} - m_{2}} \right)}{A \cdot t}$where m₁ is the mass of container with the water sample, m₂ is the massof container, A is the active membrane surface area (38.5 cm²) and t isthe time.

The macroporous gel-filled membrane may have water flux values that aresmaller than those of the unfilled support member, with possible fluxreduction of about a factor of two to about of a factor of a few hundreddepending on the application. For ultrafiltration application, the fluxmay be reduced by a factor of about ten to about a few hundred.

The hydrodynamic Darcy permeability, k (m²) of the membrane wascalculated from the following equation

$k = \frac{Q_{H_{2}O}\eta\;\delta}{3600\; d_{H_{2}O}\Delta\; P}$where η is the water viscosity (Pa·s), δ is the membrane thickness (m),d_(H20) is the water density (kg/m³), and ΔP (Pa) is the pressuredifference at which the flux, Q_(H20), was measured.

The hydrodynamic Darcy permeability of the membrane was used to estimatean average hydrodynamic radius of the pores in the porous gel. Thehydrodynamic radius, r_(h), is defined as the ratio of the pore volumeto the pore wetted surface area and can be obtained from theCarman-Kozeny equation given in the book by J. Happel and H. Brenner,Low Reynolds Number Hydrodynamics, Noordhof Int. Publ., Leyden, 1973, p.393:

$k = \frac{ɛ\; r_{h}^{2}}{K}$where K is the Kozeny constant and ε is the membrane porosity. TheKozeny constant K≈5 for porosity 0.5<ε<0.7. The porosity of the membranewas estimated from porosity of the support by subtracting the volume ofthe gel polymer.

For membrane stacks, similar flux measurements were made. As a standardprocedure, a sample in the form of a disk of diameter 4.4 cm was mountedon a sintered grid of 3-5 mm thickness and assembled into a cellsupplied with compressed nitrogen at a controlled pressure. The sampleconsisted of 1 to 4 membrane layers. In some cases layers were separatedby the mesh-spacer.

Protein Adsorption/Desorption Experiment

Protein adsorption experiments were carried out with two proteins,namely, bovine serum albumin (BSA) and lysozyme. In the case ofexperiments with a positively charged macroporous gel-filled membrane,the membrane sample was first washed with distilled water andsubsequently with a TRIS-buffer solution (pH=7.8). In an adsorptionstep, a macroporous gel-filled membrane sample in a form of a singlemembrane disk of diameter 7.8 cm was mounted on a sintered grid of 3-5mm thickness in a cell used for water flux measurements and describedabove. A BSA solution, comprising from 0.4 to 0.5 mg BSA per ml ofbuffer solution, was poured to the cell to give a 5 cm head over themacroporous gel-filled membrane. This hydrostatic pressure of 5 cm waskept constant by further additions of the BSA solution. In amodification of this method, the cell was pressurised with compressednitrogen. The flow rate was measured by weighing the amount of permeateas a function of time. Typical values varied between 1 and 5 ml/min.Permeate samples were collected at 2-5 min intervals and analyzed by UVanalysis at 280 nm. Following the adsorption step, the macroporousgel-filled membrane in the cell was washed with about 200 ml of theTRIS-buffer solution, and desorption was carried out with a TRIS-buffersolution containing 1M NaCl at 5 cm head pressure or under a controlledpressure of compressed nitrogen. The permeate samples were collected at2-5 min intervals and tested by UV analysis at 280 nm for BSA content.

For negatively charged macroporous gel-filled membranes, a solution oflysozyme in a MES buffer solution having a pH of 5.5 and a lysozymeconcentration of 0.5 g/L was used in a procedure similar to thatdescribed above for BSA and positively charged materials. The flow rateduring the protein adsorption was again kept within 1-5 ml/min. Prior tothe desorption of the protein, the membrane was washed by passing with200 ml of the buffer solution. The desorption of the protein was carriedout using a MES buffer solution (pH=5.5) containing 1 M NaCl in the sameway as described above for the desorption of BSA. The lysozyme contentin the collected samples was determined by UW spectrophotometry at 280nm.

In other examples, protein adsorption tests involve stacks of severalmembranes of diameter of 19 mm mounted into a Mustang® Coin Devicemanufactured by Pall Corporation and the protein solution was deliveredto the membrane stack at controlled flow rate using a peristaltic pump.The permeate fractions were collected and analyzed in the same way asdescribed above. The desorption of the proteins was carried in a similarway as described above, with buffered 1M NaCl delivered to the membranestack by using the pump instead of gravity or compressed nitrogenpressure.

Protein Separation Experiment

The experimental method used to examine the separation properties of theresponsive macroporous gel-filled membranes in protein-proteinfractionation processes is based on the pulsed injection ultrafiltrationtechnique and its derivatives developed by Ghosh and his co-workers anddescribed in the following articles: R. Ghosh and Z. F. Cui, Analysis ofprotein transport and polarization through membranes using pulsed sampleinjection technique, Journal of Membrane Science, vol. 175, no. 1 (2000)p. 75-84; R. Ghosh, Fractionation of biological macromolecules usingcarrier phase ultrafiltration, Biotechnology and Bioengineering, vol.74, no. 1 (2001) p. 1-11; and R. Ghosh, Y. Wan, Z. F. Cui and G. Hale,Parameter scanning ultrafiltration: rapid optimisation of proteinseparation, Biotechnology and Bioengineering, vol. 81 (2003) p. 673-682and incorporated herein by reference. The experimental set-up used wassimilar to that used for parameter scanning ultrafiltration as describedin article by R. Ghosh, Y. Wan, Z. F. Cui and G. Hale, Parameterscanning ultrafiltration: rapid optimisation of protein separation,Biotechnology and Bioengineering, vol. 81 (2003) p. 673-682.

A binary carrier phase system was used in the ultrafiltrationexperiments. The starting carrier phase in all the responsive membraneexperiments was one with a low salt concentration (typically 5-10 mMNaCl). In all these experiments the carried phase was switched to onewith a high salt concentration (typically 1 M NaCl). The change in saltconcentration within the membrane module could be tracked by observingthe conductivity of the permeate stream. The change in transmembranepressure gave an idea about the change in membrane hydraulicpermeability with change in salt concentration.

Protein adsorption experiments for the membrane stacks were carried outwith bovine serum albumin (BSA). In an adsorption step, a 4-layermacroporous gel-filled membrane sample in a form of a single membranedisk of diameter 4.4 cm was mounted on a sintered grid of 3-5 mmthickness in a cell used for water flux measurements and describedabove. A BSA solution, comprising of 0.5 mg BSA per ml of 25 mM TRISbuffer solution was poured to the cell and pressure of 35-50 kPa wasapplied. The flow rate was measured by weighing the amount of permeateas a function of time. Typical values varied between 5 and 7 ml/min.Permeate samples were collected at 2.5-3.0 min intervals and analysed byUV analysis at 280 nm.

Example 1

This example illustrates effect of pressure on the water flux throughthe 4-layers of macroporous gel-filled membrane with and withoutmesh-spacer.

A macroporous gel-filled membrane was prepared according to thefollowing procedure:

A solution containing 2.14 g of (3-acrylamidopropyl)-trimethylammoniumchloride (APTAC) monomer as a 75% aqueous solution, 0.06 g ofN,N′-methylenebisacrylamide (BIS) as a cross-linker, and 0.02 g ofIrgacure® 2959 as a photoinitiator dissolved in 8.75 g of di(propyleneglycol)methylether (DPM) was prepared.

A macroporous gel-filled membrane was prepared from the solution and thesupport TR2611A using the photoinitiated polymerization according to thegeneral procedure described above. The irradiation time used was 5minutes at 350 nm. The macroporous gel-filled membrane was removed frombetween the polyethylene sheets, washed with water and TRIS-buffersolution.

Several samples similar to that described above were prepared andaveraged to estimate the mass gain of the macroporous gel-filledmembrane. The substrate gained 98.5% of the original weight in thistreatment.

The single layer macroporous gel-filled membrane produced by this methodhad a water flux in the range of 1890 kg/m² hr at 100 kPa. A 4-layersmembrane stack with and without spacers was tested for the water fluxthrough it.

FIG. 6 shows effect of pressure on the water flux of the 4-layersmembrane stack with and without spacers.

As can be seen from FIG. 6, stacks of these membranes exhibit anon-linear pressure flux relationship. In the non-mesh-spacer case, theflux reaches a limiting value as the pressure is increased.

Example 2

This example illustrates effect of increasing number of layers in amembrane stack on the water flux through the macroporous gel-filledmembrane with and without mesh-spacer.

A macroporous gel-filled membrane was prepared and tested for the waterflux according to procedures as described above.

FIG. 7 illustrates the effect of increasing the number of layers in astack with and without interleaving mesh-spacers on the water flux.

These data again show that we are achieving a substantial improvement influx by using the interleaving approach.

Example 3

This example illustrates effect of mesh-spacer on the sharpnessbreakthrough BSA adsorption curve for 4-layers Q-type macroporousgel-filled membrane.

A macroporous gel-filled membrane was prepared according to procedure asdescribed above.

The protein (BSA) adsorption characteristic of the macroporousgel-filled membrane was examined using the general procedure for amulti-layer membrane disk as described above. The concentration of theprotein used in this experiment was 0.5 g/L in 25 mM TRIS-buffer. Theflow rate was 5-7 ml/min. A plot of the concentration of BSA in thepermeate vs. the permeate volume is shown in FIG. 8. The macroporousgel-filled membrane had a BSA binding capacity of 200 mg/ml.

It could be expected that insertion of the interleaving layers couldlead to a loss of resolution of the stacks in a separation. Surprisinglythis is not the case. As is shown in FIG. 8 the breakthrough curve of afour membrane stack with interleaving mesh layers is in fact somewhatsharper than the four layers without interleaving. This invention thusnot only improves substantially the flux of a stack at a given pressurebut also improves its performance in a separation.

Example 4

This example illustrates the formation of an unsupported porous gel,which can be used as the macroporous gel to prepare the macroporousgel-filled membrane.

A solution containing 3.33 g of (3-acrylamidopropane)trimethylammoniumchloride (APTAC) monomer as a 75% aqueous solution, 0.373 g ofN,N′-methylenebisacrylamide (BIS) cross-linker, and 0.0325 g ofIrgacure® 2959 photoinitiator dissolved in 25 ml of adioxane:dimethylformamide:water mixture, with the solvent volume ratioof 71:12:17, respectively, was prepared. In this solvent mixture,dioxane is a poor solvent while DMF and water are good solvents. A totalmonomer concentration (APTAC and BIS) of 0.58 mol/L was thus obtained.The cross-linking degree was 20 mol %, based on APTAC. 5 ml of thissolution was placed in a glass vial and subjected to UV irradiation at350 nm for 2 hrs. A white gel was formed which was washed thoroughlywith de-ionized water to exchange the reaction solvent and remove theunreacted monomer or soluble oligomers.

The gel formed was mechanically very weak. A sample of the gel wasexamined using an environmental scanning electron microscope (ESEM) withwater vapor present in the sample chamber to prevent drying of the gel.The micrograph, shown in FIG. 9, has dark, cavernous areas that indicatethat a macroporous gel was formed.

Example 5

This example illustrates a method of preparing a positively chargedmacroporous gel-filled membrane using the monomer solution ofcomposition described in example 4 applied to a sample of thepoly(propylene) porous support PP1545-4. The macroporous gel-filledmembrane was prepared according to the general procedure described aboveusing UV irradiation at 350 nm for 2 hours. After polymerization, themacroporous gel-filled membrane was washed with with de-ionized waterfor 48 hrs.

Mass gain of the resulting macroporous gel-filled membrane after dryingwas 107 wt %, water flux was 1643±5 kg/m²h at 50 kPa and Darcypermeability was 9.53×10⁻¹⁶ m².

The morphology of the gel-incorporated macroporous gel-filled membranewas examined using ESEM in the same manner as described in Example 4.The ESEM micrograph shown in FIG. 10 shows that the macroporous gel hasbeen incorporated into the host membrane. The micrograph shows a similarstructure to that of the unsupported macroporous gel shown in FIG. 9 andlittle evidence of the microporous support member.

Example 6

This example illustrates a method of preparing a negatively chargedmacroporous gel-filled membrane, with a weak acid functionality.

5.50 g of vacuum-distilled methacrylic acid (MAA) monomer, 0.4925 g ofN,N′-methylenebisacrylamide cross-linker and 0.1503 g of Irgacure® 2959photoinitiator were dissolved in 25 ml of a dioxane:DMF solvent mixturewith a volume ratio of 9:1, respectively, to prepare the startingmonomer solution. The macroporous gel-filled membrane was prepared usingthe poly(propylene) PP1545-4 support and the general procedure for thephotoinitiated polymerization described above. The irradiation time usedwas 2 hours and the resulting membrane was washed with DMF for 24 hrsfollowed by a 48 hr wash with deionized water. The mass gain of theresulting dried membrane was 231 wt %, water flux was 4276±40 kg/m²h at50 kPa and Darcy permeability was 2.64×10⁻¹⁵ m².

The protein (lysozyme) absorption/desorption characteristics of themacroporous gel-filled membrane were examined using the generalprocedure for a single membrane disk outlined earlier. The concentrationof the protein used in this experiment was 0.5 g/L in a 10 mM MES bufferat pH 5.5. The flow rate of adsorption experiment was regulated to be2-4 ml/min. A plot of the concentration of lysozyme in permeate versusthe volume of permeate is shown in FIG. 11. It can be seen that evenwith the single membrane disk, a relatively steep break through curve isobtained indicating a uniform and narrow pore size distribution in themembrane. The macroporous gel-filled membrane has a breakthroughlysozyme binding capacity of 42.8 mg/mL. A desorption experiment with abuffer solution containing 1M NaCl indicated that the recovery ofprotein was 83.4%.

Example 7

This example illustrates the effect of the total monomer concentrationand solvent mixture on the hydraulic flow rate (flux) of compositemembranes with weak acid functionality of the type described in Example3.

A series of composite membranes (MAA1 through MAA5) were prepared usingmonomer solutions of chemical compositions listed in Table 1 and theporous support PP1545-4. The preparation procedure described in Example3 was employed.

TABLE 2 The effect of the total monomer concentration and solventmixture on water flux of composite membranes Total Monomer ConcentrationCrosslinking Solvent Mixture Mass Flux at Sample (MAA + BIS) Degree(volume part) Gain 50 kPa I.D. (mol/L) (mol-%) Dioxane DMF (wt %) (kg/m²· h) MAA1 1.71 5 8 2 71 12.2 ± 0.1 MAA2 2.19 5 8 2 153  94 ± 14 MAA32.68 5 8 2 177 1265 ± 111 MAA4 3.66 5 8 2 300 1800 ± 9  MAA5 2.68 5 9 1231 4276 ± 40 

As can be seen from Table 2, the hydraulic flow rate (flux) of compositemembranes can be tuned by adjusting the monomer loading in the solution.Contrary to the typical trends found with homogeneous gels, for which anincrease in gel density is followed by decrease in permeability, theincrease in the mass gain in the membranes of this series results in theflux increase. Further increase in flux is achieved when theconcentration of the poor solvent (dioxane) in the solvent mixture isincreased (compare samples MAA3 and MAA5).

Example 8

This example illustrates a method of preparing a negatively chargedmacroporous gel-filled membrane that has strong acid functionality.

A solution containing 2.50 g 2-acrylamido-2-methyl-1-propanesulfonicacid (AMPS) monomer, 0.372 g N,N′-methylenebisacrylamide cross-linkerand 0.0353 g Irgacure® 2959 photo-initiator, dissolved in 25 ml of adioxane:H₂O mixture with a volume ratio 9:1, respectively, was used. Amacroporous gel-filled membrane was prepared from the solution and thesupport PP1545-4 using the photoinitiated polymerization according tothe general procedure describe above. The irradiation time used was 1hour at 350 nm. After polymerization, the membrane was extracted withde-ionized water for 48 hrs. The mass gain of the resulting membrane was74.0 wt %, water flux was 2559±40 kg/m²h at 50 kPa and Darcypermeability was 1.58×10⁻¹⁵ m².

Example 9

This example illustrates further the effect of the solvent mixturecomposition and the cross-linking degree on the hydraulic flow rate ofcomposite membranes with the strong acid functionality. A series ofcomposite membranes (AMPS1 through AMPS5) was prepared using chemicalcompositions listed in Table 3 following the general preparationprocedure and the irradiation conditions as in example 5.

TABLE 3 The effect of solvent mixture on water flux of the compositemembranes Total Monomer Crosslinking Solvent mixture Mass Flux at SampleConc. Degree (volume part) gain 50 kPa I.D. (mol/L) (mol %) Dioxane DMFH₂O (wt %) (kg/m²h) AMPS1 0.48 20 5 5 0 92 3.2 ± 0.0 AMPS2 0.48 20 8 2 0100 575 ± 12  AMPS3* 0.48 20 9 0 1 74 2559 ± 9   AMPS4 0.48 10 8 2 0 1008.4 ± 0.0 AMPS3 is the composite membrane prepared in the previousExample.

AMPS3 is the composite membrane prepared in the previous Example.

As can be seen, a similar pattern to that described in example 7 wasobserved with regards to the relationship between solubility of polymerin the solvent and water flux of composite membranes. Comparison ofAMPS2 with AMPS 4 shows that hydraulic flow rate (flux) of a compositemembrane can also be adjusted by the degree of cross-linking.

Example 10

This example illustrates the effect of introducing a neutral co-monomerinto a negatively charged macroporous gel-filled membrane.

A solution containing 1.750 g of 2-acrylamido-2-methyl-1-propanesulfonicacid, 0.485 g of acrylamide, 0.868 g of N,N′-methylenebisacrylamidecross-linker, and 0.044 g of Irgacure® 2959 photo-initiator, dissolvedin 25 ml of a dioxane:DMF:H₂O mixture with a volume ratio 8:1:1,respectively, was prepared. A macroporous gel-filled membrane wasprepared from the solution and the support PP1545-4 using thephotoinitiated polymerization according to the general proceduredescribe above. The irradiation time used was 1 hour at 350 nm. Afterpolymerization, the membrane was extracted with de-ionized water for 48hrs.

The mass gain of the resulting membrane was 103 wt %, water flux was7132±73 kg/m²·h at 100 kPa, and Darcy permeability was 4.40×10⁻¹⁵ m².

Example 11

This example illustrates one method of making a positively chargedmacroporous gel-filled membrane.

A 15 wt-% solution was prepared by dissolving diallyldimethylammoniumchloride (DADMAC) monomer and N,N′-methylenebisacrylamide (BIS)cross-linker in a molar ratio of 5:1, respectively, in a solvent mixturecontaining 37 wt-% water, 45 wt-% dioxane and 18 wt-% DMF. Thephoto-initiator Irgacure® 2959 was added in the amount of 1% withrespect to the mass of the monomers.

A macroporous gel-filled membrane was prepared from the solution and thesupport PP1545-4 using the photoinitiated polymerization according tothe general procedure describe above. The irradiation time used was 30minutes at 350 nm. The macroporous gel-filled membrane was removed frombetween the polyethylene sheets, washed with water and TRIS-buffersolution and stored in water for 24 hrs.

Several samples similar to that described above were prepared andaveraged to estimate the mass gain of the macroporous gel-filledmembrane. The substrate gained 42.2% of the original weight in thistreatment.

The macroporous gel-filled membrane produced by this method had a waterflux in the range of 2100-2300 kg/m² hr at 70 kPa and Darcy permeabilityof 9.87×10⁻¹⁶ m².

The protein (BSA) adsorption characteristic of the macroporousgel-filled membrane was examined using the general procedure for asingle membrane disk described above. The concentration of the proteinused in this experiment was 0.4 g/L in 50 mM TRIS-buffer. The flow ratewas 2-4 ml/min. A plot of the concentration of BSA in the permeate vs.the permeate volume is shown in FIG. 12. The macroporous gel-filledmembrane had a BSA binding capacity of 48-51 mg/ml. The BSA desorptionwas found to be in the range of 78-85%.

Example 12

This example illustrates that by adding a neutral monomer to the chargedmonomer used in Example 9 the protein binding capacity can besubstantially increased.

A 10 wt-% solution was prepared by dissolving diallyldimethylammoniumchloride (DADMAC) and acrylamide (AAM), in the ratio 80:20, in a solventmixture containing 63 wt-% dioxane, 18 wt-% water, 15 wt-% DMF, and 4wt-% dodecanol. N,N′-methylenebisacrylamide cross-linker was added tothe monomer solution to obtain 40% (mol/mol) cross-linking degree. Thephotoinitiator Irgacure® 2959 was added in the amount of 1% with respectto the total mass of monomers.

A macroporous gel-filled membrane was prepared from the solution and thesupport PP1545-4 using the photoinitiated polymerization according tothe general procedure describe above. The irradiation time used was 20minutes at 350 nm. The macroporous gel-filled membrane was removed frombetween the polyethylene sheets, washed with water, TRIS-buffer solutionand stored in water for 24 hrs.

A similar sample to that described above was prepared and used toestimate the mass gain of the macroporous gel-filled membrane. Thesubstrate gained 80% of the original weight in this treatment.

The macroporous gel-filled membrane produced by this method had a waterflux in the range of 250 kg/m² hr at 70 kPa and Darcy permeability was1.09×10⁻¹⁶ m².

The protein (BSA) adsorption characteristic of the macroporousgel-filled membrane was examined using the general procedure for asingle membrane disk described above. The protein concentration was 0.4g/L in a 50 mM TRIS buffer solution. The flow rate of absorptionexperiment was adjusted to 2-4 ml/min. The macroporous gel-filledmembrane had a BSA binding capacity of 104 mg/ml.

Example 13

This example illustrates the formation of a supported porous gelmacroporous gel-filled membrane by cross-linking of a pre-formedpolymer.

Three separate solutions were prepared with the following compositions:(A) 20 g of branched poly(ethyleneimine) (BPEI) (25,000 Da) in 50 ml ofmethanol, (B) 20 g of poly(ethyleneglycol) PEG (˜10,000 Da) in 50 ml ofmethanol, and (C) ethyleneglycol diglycidyl ether (0.324 g) in 5 ml ofmethanol.

A mixture of the three solutions was prepared consisting of 2 ml of (A),3 ml of (B), and 5 ml of (C). A portion of this resulting solution wasallowed to stand in a vial overnight when a phase separation wasobserved. Examination of the morphology of the upper clear gel layerindicated that it was macroporous.

The same mixed solution was spread on a sample of poly(propylene)support PP1545-4 using the techniques described in the generalprocedure. The membrane was sandwiched between twopoly(ethyleneterephthalate) sheets and allowed to stand overnight. Themacroporous gel-filled membrane was extracted with methanol at roomtemperature for 24 h, and a mass gain of 95% was observed. The waterflux of the macroporous gel-filled membrane was 6194 kg/m² h at 100 kPaand Darcy permeability was 4.89×10⁻¹⁰ m².

The dynamic protein absorption capacity of the macroporous gel-filledmembrane was measured using a BSA solution (0.4 mg/mL) in the method fora single membrane disk described in the general section above. It had acapacity of 68 mg/ml before breakthrough.

Example 14

This example illustrates the effect of monomer mixture composition andthe polymerization conditions on the hydraulic properties of macroporousgel-filled membranes prepared by in situ polymerization of glycidylmethacrylate (GMA) with ethylene dimethacrylate (EDMA) used as across-linker. The solvents used were dodecanol (DDC), cyclohexanol(CHX), and methanol. A porous polypropylene support membrane PP1545-4and two modes of initiation of in situ polymerization were usedaccording to the general procedure described above. In thephotopolymerization mode, 2,2-dimethoxy-2-phenylacetophenone (DMPA) wasused as a photoinitiator while the thermal polymerization was initiatedby 1,1′-azobis(cyclohexanecarbonitrile). In both modes, thepolymerization was carried out for 2 hours.

The polymerization conditions and properties of the macroporousgel-filled membranes containing porous poly(glycidylmethacrylate-co-ethylene diacrylate) are presented in Table 4.

TABLE 4 Porous poly(glycidyl methacrylate-co-ethylene diacrylate)-filledmacroporous gel-filled membranes Total Monomer Initiation Mode MassDarcy Hydrodynamic Membrane Concentration of Gain Permeability radius IDwt-% Solvent Polymerization wt-% m² nm AM612 43.8 DDC/CHX 9/91 Photo276.8 6.96 × 10⁻¹⁶ 95.1 AM614 22.9 DDC/CHX 9/91 Photo 144.3 2.77 × 10⁻¹⁵148.5 AM615 47.6 DDC/CHX 9/91 Thermal 237.0 1.66 × 10⁻¹⁷ 13.0 AM616 24.9DDC/CHX 9/91 Thermal 157.5 9.15 × 10⁻¹⁶ 86.4 AM619 48.6 Methanol Photo265.0 2.48 × 10⁻¹⁵ 163.8

The mass gain obtained in this series of macroporous gel-filledmembranes is proportional to the total monomer concentration in thepolymerization mixture. Membranes AM612, AM615, and AM619 were preparedusing high concentration of monomers while in membranes AM614 and AM616the monomer concentration was cut approximately by half (Table 4).

The high values of the pure water flux measured at 100 kPa oftransmembrane pressure (Table 4) indicate that the pore-filling materialis macroporous. The pure water flux and, consequently, the hydraulicradius are affected not only by the mass gain but also by thepolymerization mode. As shown in FIG. 13, the hydraulic radius is alinear function of the mass gain with the slope depending on thepolymerization mode. The absolute value of the negative slope in thethermal polymerization is twice that of the photopolymerized macroporousgel-filled membranes. This means that photopolymerized macroporousgel-filled membranes have larger pores than that of the thermallypolymerized ones at the same mass gains. Thus, the photo-initiatedpolymerization, which is faster than the thermally-initiated one,produces larger pores. Since the monomer conversion is practically thesame in both cases of polymerization (similar mass gains), the presenceof the poly(propylene) substrate either through its hydrophobic natureor by creating microscopic confinements for the polymerization affectsthe pore formation and the final structure of the pore-fillingmaterials.

By changing the solvents from dodecanol/cyclohexanol 9/91 to methanol,which is cheaper and environmentally more acceptable than the othersolvents, a macroporous gel-filled membrane with very high flux wasobtained (membrane AM619, Table 4). The macroporous gel-filled membranewas produced from the concentrated monomer mixture and had fluxcomparable with that of the membrane AM614 which had a mass gain almosttwice as low as that of AM619.

This and subsequent examples illustrate a feature of some macroporousgel-filled membranes. With the capability to change the composition andconcentration of the monomers and solvents, there can be produced stablemacroporous gel-filled membranes with different porous structures. Asshown in Table 4, macroporous gel-filled membranes with larger pores canbe made in this way.

Example 15

This example illustrates further the effect of monomer mixturecomposition on the hydraulic properties of macroporous gel-filledmembranes.

A series of macroporous gel-filled membranes have been preparedaccording to the general procedure described above and containing porouspoly(acrylamide) gels formed by in situ photoinitiated polymerization ofacrylamide (AAM) and N,N′-methylenebisacrylamide (BIS) as a cross-linkerin the pores of a poly(propylene) support membrane. The porous supportmember used was poly(propylene) TIPS membrane PP1545-4.2,2-Dimethoxy-2-phenylacetophenone (DMPA) or1-[4-(2-hydroxyethoxy)phenyl]2-hydroxy-2-methyl-1-propane-1-one(Irgacure® 2959) were used as photoinitiators. Irradiation was carriedout at 350 nm for 2 hours. Composition of the pore-filling solutions andthe properties of the resulting macroporous gel-filled membranes aresummarized in Table 5.

TABLE 5 Composition and properties of poly(acrylamide)-filledmacroporous gel-filled membranes Total Degree Monomer Solvent 1 Solvent2 Darcy Hydrodynamic Membrane of XL Conc. Conc. Conc. Mass PermeabilityRadius I.D. Wt-% wt-% Name wt-% Name wt-% Gain % m² nm AM606 18.0 13.3Water 86.6 None 0.0 111.7 9.3 × 10⁻¹⁸ 8.0 AM607 18.0 13.6 Water 67.9Methanol 18.4 107.8 2.5 × 10⁻¹⁸ 4.1 AM608 18.0 14.4 Water 71.8 Glycerol13.7 110.4 2.1 × 10⁻¹⁸ 3.8 AM609 16.8 12.0 Water 51.9 Glycerol 36.0103.3 1.6 × 10⁻¹⁸ 3.3 AM610 31.8 34.5 DMF 49.1 1-Propanol 16.4 307.2 3.9× 10⁻¹⁷ 20.0 AM611 32.0 18.7 DMF 60.9 1-Propanol 20.5 130.3 5.3 × 10⁻¹⁶62.7 AM617 32.2 34.9 DMF 48.4 1-Octanol 16.7 273.3 8.6 × 10⁻¹⁷ 29.0

Membranes AM606 through AM609 have been prepared using very similarconcentration of monomers (12.0-14.4 wt-%) and a similar, relativelyhigh, degree of cross-linking (16.8-18.0 wt-% of monomers). The massgains obtained with these macroporous gel-filled membranes are also verysimilar. As shown in FIG. 14, there is a linear relationship betweentotal monomer concentration in the pore-filling solution and the massgain achieved after photopolymerization.

The high degree of cross-linking in the macroporous gel-filled membraneprepared from aqueous solution without non-solvent (AM606) leads torelatively high permeability. Surprisingly, the addition of methanol orglycerol, which are poor solvents for linear poly(acrylamide), to water,which is a good solvent for the linear polymer, brings about asubstantial reduction in the Darcy permeability and the hydrodynamicradius calculated on its basis. The reduction in permeability is higherwith glycerol than with methanol and increases with the amount ofglycerol in the solution.

The use of mixtures of poor solvents, such as N,N′-dimethylformamide and1-propanol or 1-octanol, as well as the further increase of the degreeof cross-linking and total monomer concentration have been tested inmembranes AM610, AM611, and AM617. As shown in Table 5, substantiallyhigher permeabilities and hydraulic radii are obtained with all thesemacroporous gel-filled membranes as compared to the macroporousgel-filled membranes prepared with water as one of the solvents. Thisoccurred despite an increase of the total monomer concentration; morethan double in membranes AM610 and AM617 than that in the membranesprepared with water as one of the solvents. Changing the other solventfrom 1-propanol in AM610 to 1-octanol in AM617 also brings aboutsubstantial increase in permeability and hydraulic radius.

Microscopic images of the surface of membrane AM610 are shown in FIGS.15 (AFM) and 16 (ESEM). For comparison, an ESEM image of the nascentporous support member is also shown in FIG. 16. Both sets of images showa porous phase-separated gel covering the member surface with nodiscernible elements of the support member.

Membrane AM611 was prepared with DMF and 1-propanol but the totalmonomer concentration was just over half that of AM610. Membrane AM611shows very high flux and the hydraulic radius three times that of AM610.The ESEM images of the surface of this membrane are presented in FIG.17. It shows a highly porous gel structure (top image) that resemblesthe bulk gel formed in some spots on the membrane surface but detachedfrom the membrane (bottom picture).

A comparison of surfaces of membranes AM610 and AM611 is presented inFIG. 18. The large difference in the size of the structural elements inthese two gels is clearly visible.

The macroporous gel-filled membranes prepared in this example can serveas ultrafiltration membranes. It has been shown that the pore size ofthe macroporous gel-filled membrane and, therefore, its separationproperties, can be controlled to achieve a wide range of values.

Example 16

This example illustrates the effect of pore size of the support memberon the hydraulic flow rate (flux) through macroporous gel-filledmembranes.

Two polypropylene support membranes of pore size 0.45 μm and 0.9 μm,PP1545-4 and PP1183-3X, respectively, were used to produce macroporousgel-filled membranes with the same monomer mixture containing 39.4 wt-%of glycidyl methacrylate and 9.2 wt-% of ethylene diacrylate inmethanol, thus having 48.6 wt-% of monomers and 18.9 wt-% of ethylenediacrylate (cross-linker) in the monomer mixture. The photoinitiatorused was DMPA in the amount of 1.3 wt-% of monomers.

The macroporous gel-filled membranes were prepared according to thegeneral procedure described above. The irradiation time was 2 hours at350 nm. The resulting macroporous gel-filled membranes were washed withmethanol followed by deionized water. The macroporous gel-filledmembranes were tested for water flux at 100 kPa to calculate the Darcypermeability and hydraulic radius. The results are presented in Table 6.

TABLE 6 Hydraulic properties of composite membranes produced withsubstrates of different pore sizes Average Standard Membrane Supportpore Mass Gain Flux at 100 Hydrodynamic hydrodynamic Deviation ID size(μm) (wt-%) kPa (kg/m²h) radius (nm) radius (nm) (%) AM619 0.45 265.08080.9 163.8 156.2 6.9 AM620 0.90 296.8 7310.1 148.5

The data shows that the hydraulic radius in both macroporous gel-filledmembranes is the same within an experimental error, proving that themacroporous gel-filled membranes contain macroporous gels of similarstructure.

Example 17

This Example illustrates the synthesis of poly(ethylene glycol) (PEG,MW's 4000, 2000, 1,000, and 200) diacrylates, which can be used ascross-linkers to prepare the macroporous gel-filled membrane.

The synthesis procedure used follows that described by N. Ch.Padmavathi, P. R. Chatterji, Macromolecules, 1996, 29, 1976, which isincorporated herein by reference. 40 g of PEG 4000 was dissolved in 150ml of CH₂Cl₂ in a 250-ml round bottom flask. 2.02 g of triethylamine and3.64 g of acryloyl chloride were added dropwise to the flask separately.Initially the reaction temperature was controlled at 0° C. with an icebath for 3 hrs, and then the reaction was allowed to warm to roomtemperature and kept for 12 hrs. The reaction mixture was filtered toremove the precipitated triethylamine hydrochloride salt. The filtratethen was poured into an excess of n-hexane. The colorless product,referred to as PEG 4000 diacrylate, was obtained by filtration anddrying at room temperature.

The same procedure was used with the PEG's of other molecular weights.The molar ratios of the PEG to acryloyl chloride were kept the same asused above with PEG 4000.

Example 18

This example illustrates a further method of preparing a negativelycharged macroporous gel-filled membrane that has a high adsorptioncapacity for lysozyme.

A solution containing 0.6 g of 2-acrylamido-2-methyl-1-propanesulfonicacid (AMPS), and 0.4 g of acrylamide (AAM) as monomers, 0.25 g ofN,N′-methylenebisacrylamide (BIS) and 1.0 g of PEG 4000 diacrylateobtained in Example 14 as cross-linkers, and 0.01 g of Iragure® 2959 asa photoinitiator was prepared in 10 ml of solvent consisting of a80:10:10 volume ratio of dioxane, dimethylformamide (DMF), and water.

A porous poly(propylene) support member in the form of a membrane(PP1545-4) was used and the macroporous gel-filled membrane was preparedaccording to the general procedure, with the irradiation carried out at350 nm for 20 minutes. After polymerization, the macroporous gel-filledmembrane was washed thoroughly with de-ionized water for 24 hrs.

Mass gain of the resulting macroporous gel-filled membrane after dryingwas 113.2 wt %, water flux was 366±22 kg/m²h at 100 kPa, and Darcypermeability was 2.26×10⁻¹⁶.

The protein (lysozyme) absorption/desorption characteristics of themacroporous gel-filled membrane were examined using the generalprocedure for a single membrane disk outlined earlier. The concentrationof the protein used in this experiment was 0.5 g/L in a 10 mM MES bufferat pH 5.5. The flow rate of adsorption experiment was regulated to be2-4 ml/min. A plot of the concentration of lysozyme in the permeateversus the volume of permeate is shown in FIG. 19. It can be seen that arelatively steep break through curve is obtained. The macroporousgel-filled membrane had a Lysozyme binding capacity of 103.9 mg/ml. Adesorption experiment indicated that the recovery of protein was 64.0%.

Example 19

This example illustrates preparation of a negatively charged macroporousgel-filled membrane with the same nominal polymer composition as inExample 18 but with much higher hydraulic flows (flux) and good lysozymeuptake capacity.

The monomer solution was produced by dilution of the solution formulatedin Example 15 with acetone with the mass ratio of 1:1.

A porous poly(propylene) support member in the form of a membrane(PP1545-4) was used and the macroporous gel-filled membrane was preparedaccording to the general procedure. The irradiation time used was 2hours. After polymerization, the macroporous gel-filled membrane waswashed thoroughly with de-ionized water for 24 hrs.

The mass gain of the resulting macroporous gel-filled membrane afterdrying was 51.1 wt % and water flux was 6039±111 kg/m²·h at 100 kPagiving Darcy permeability of 3.73×10⁻¹⁵.

The protein (lysozyme) absorption/desorption characteristics of themacroporous gel-filled membrane were examined using the generalprocedure for a single membrane disk outlined earlier. The concentrationof the protein used in this experiment was 0.5 g/L in a 10 mM MES bufferat pH 5.5. The flow rate of adsorption experiment was regulated to be2-4 ml/min. A plot of the concentration of lysozyme in permeate versusthe volume of permeate is shown in FIG. 20. It can be seen that arelatively steep break through curve is obtained. The macroporousgel-filled membrane had a lysozyme binding capacity of 75.4 mg/ml. Adesorption experiment indicated that the recovery of protein was 65.0%.

Example 20

This example illustrates a further preparation of a negatively chargedmacroporous gel-filled membrane that has a very high flux but lowerprotein binding capacity.

The monomer solution was produced by dilution of the solution formulatedin Example 15 with acetone with the mass ratio of 1:2.

A porous poly(propylene) support member in the form of a membrane(PP1545-4) was used and the preparation of macroporous gel-filledmembrane was carried out according to the general procedure describedabove. UV initiated polymerization was carried out for 2 hours. Afterpolymerization, the macroporous gel-filled membrane was washedthoroughly with de-ionized water for 24 hrs.

The mass gain of the resulting macroporous gel-filled membrane afterdrying was 34.4 wt % and water flux was 12184±305 kg/m²h at 100 kPagiving Darcy permeability of 7.52×10⁻¹⁵.

The protein (lysozyme) absorption/desorption characteristics of themacroporous gel-filled membrane were examined using the generalprocedure for a single membrane disk outlined earlier. (Theconcentration of the protein used in this experiment was 0.5 g/L in a 10mM MES buffer at pH 5.5. The flow rate of adsorption experiment wasregulated to be 2-4 ml/min.) A plot of the concentration of lysozyme inpermeate versus the volume of permeate is shown in FIG. 21. It can beseen that a relatively steep break through curve is obtained. Themacroporous gel-filled membrane had a lysozyme binding capacity of 53.5mg/ml. A desorption experiment indicated that the recovery of proteinwas 99.0%.

Examples 18, 19 and 20 show that it is possible to control the loadingof porous gel into the host membrane thereby controlling the water fluxat a defined pressure (100 kPa in the data given in the examples) andalso that the lysozyme uptake is related to the mass of incorporatedporous gel.

Example 21

This example illustrates preparation of a negatively charged membranethat has both good protein adsorption capacity and good flux using amacromonomer.

A monomer solution containing 0.6 g of2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 0.4 g of acrylamide(AAM), 0.25 g of N,N′-methylenebisacrylamide (BIS), 0.01 g of Irgacure®2959, and 1.0 g of PEG 2000 macromonomer obtained in Example 14,dissolved in 10 ml of a dioxane-(DMF)-water mixture with a volume ratio80:10:10, respectively, was prepared.

A microporous poly(propylene) support member in the form of a membrane,support PP1545-4, was used together with the general procedure describedabove. The irradiation time used was 20 minutes. After polymerization,the membrane was washed thoroughly with de-ionized water for 24 hrs.

The mass gain of the resulting membrane after drying was 108.4 wt % andwater flux was 1048±4 kg/m²h at 100 kPa giving Darcy permeability of6.47×10⁻¹⁶.

The protein (lysozyme) adsorption/desorption characteristics of themembrane were examined using the general procedure for a single membranedisk outlined earlier. A relatively steep break through curve wasobtained. The membrane had a lysozyme binding capacity of 88.7 mg/ml.The desorption experiment indicated that the recovery of protein was64.0%.

Example 22

This example in combination with example 18 above further illustratesthat the protein binding capacity and flow characteristics of a membranecan be tuned.

The monomer solution was produced by dilution of the solution formulatedin Example 21 with acetone with the mass ratio of 1:1.

A porous poly(propylene) support member in the form of a membrane,support PP1545-4, was used along with the general procedure for thepreparation of macroporous gel-filled membranes described above. Theirradiation time used was 90 minutes. After polymerization, the membranewas washed thoroughly with de-ionized water for 24 hrs.

The mass gain of the resulting membrane after drying was 45.7 wt % andwater flux was 7319±180 kg/m h at 100 kPa.

The protein (lysozyme) absorption/desorption characteristics of themembrane were examined using the general procedure for a single membranedisk outlined earlier. A relatively steep break through curve wasobserved. The membrane had a lysozyme binding capacity of 63.4 mg/ml.The desorption experiment indicated that the recovery of protein was79.3%.

Example 23

This example illustrates the effect of a neutral co-monomer on theprotein binding capacity of macroporous gel-filled membranes.

TABLE 7 Chemical composition of stock solutions (amount of monomers in10 mL of a solution) Solvents (Dioxane/DMF/H₂O) Stock AAM AMPS PEG2000XLBIS Irgacure ® volumetric ID (g) (g) (g) (g) 2959, (g) ratio S1 0.600.40 1.00 0.25 0.01 8:1:1 S2 0.40 0.60 1.00 0.25 0.01 8:1:1 S3 0.20 0.801.00 0.25 0.01 8:1:1 S4 0 1.00 1.00 0.25 0.01 8:1:1 PEG2000XL: PEG2000diacrylate prepared in example 14

Monomer solutions were prepared by dilution of stock solutions S1-S4 inTable 7 with acetone with the mass ratio of 1:1.

Composite membranes M1-M4 were prepared by using the correspondingdiluted solutions of stocks S1-S4 and following the general preparationprocedure described earlier. The porous support used was PP1545-4 andthe irradiation time was 90 minutes. Upon completion of polymerization,the composite membranes were washed with de-ionized water for 24 hrs.

The properties and protein binding capacities of composite membraneswere examined and the results shown in Table 8. It is evident that thecharge density of polyelectrolyte gels influences significantly proteinadsorption onto membranes.

TABLE 8 Properties and Lysozyme adsorption capacities of compositemembranes Flux at Binding 100 kPa Capacity No. (kg/m² · h) (mg/ml) M18146 ± 96 56.9 M2 4273 ± 46 76.3 M3 7940 ± 303 41.5 M4 8651 ± 72 16.4

Example 24

This example illustrates the effect of the chain length of thepolyfunctional macromonomers used as cross-linkers (PEG diacrylates) onprotein binding capacity of macroporous gel-filled membranes.

A series of stock solutions containing 0.6 g of2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 0.4 g of acrylamide(AAM), 0.10 g of N,N′-methylenebisacrylamide (BIS), 0.01 g of Irgacure®2959, and 1.0 g of PEG diacrylate with different molecular weights (200,1000, 2000, 4000), obtained in Example 17, dissolved in 10 ml of adioxane-DMF-water mixture with a volume ratio 80:10:10, respectively,was prepared. The stock solutions were subsequently diluted with acetoneat the mass ratio of 1:1. A series of composite membranes were preparedfrom these solutions using poly(propylene) support PP1545-4 and byfollowing the general preparation procedure described above. Theirradiation time used was set to 90 minutes. Upon completion ofpolymerization, the composite membranes were washed with de-ionizedwater for 24 hrs.

The properties and protein binding capacities of composite membraneswere examined according to the general procedure for a single membranedisk. The results shown in Table 9 clearly indicate that the gelstructure of composite membranes has substantial effect on proteinadsorption. Possibly, it is related to the gel structure near themacropore surface, where an extremely loose structure may be formed thatcan allow protein to penetrate into the gel layer at a certain depth.Another possibility is that by using a longer chain PEG diacrylate thesurface area is increased owing to some fuzziness at the surface andthus making more adsorption site available to proteins.

TABLE 9 Properties and Lysozyme adsorption capacities of compositemembranes Flux at Binding PEG 100 kPa Capacity diacrylate (kg/m²h)(mg/ml) 200 8390 ± 218 24.4 1000 7275 ± 139 58.1 2000 4273 ± 46 76.34000 6039 ± 111 75.4

Example 25

This example illustrates the use of fibrous non-woven support to producea macroporous gel-filled membrane containing positively chargedmacroporous gel.

A 10 wt-% solution was prepared by dissolving diallyldimethylammoniumchloride (DADMAC) and acrylamide (AAM), which were taken in the ratio80:20, in a solvent mixture containing 65 wt-% of dioxane, 18 wt-% ofwater, and 17 wt-% of DMF. N,N′-methylenebisacrylamide (BIS) was addedto the monomer solution to obtain 40% (mol/mol) cross-linking degree.The photoinitiator Irgacure® 2959 was added in the amount of 1% withrespect to the total mass of the monomers.

A sample of the fibrous non-woven polypropylene substrate TR2611A wasplaced on a polyethylene sheet and filled with the monomer solution. Thesubstrate was subsequently covered with another polyethylene sheet andthe resulting sandwich was run between two rubber rollers to press themonomer solution into the pores and remove excess of solution. Thefilled substrate was irradiated at 350 nm for 20 min for thepolymerization process to occur. The macroporous gel-filled membrane wasremoved from between the polyethylene sheets, washed with water,TRIS-buffer solution and stored in water for 24 hrs. A duplicate samplewas used to estimate the mass gain of the macroporous gel-filledmembrane. The substrate gained 45% of the original weight in thistreatment.

The macroporous gel-filled membrane produced by this method had a waterflux in the range of 2320 kg/m² hr at 70 kPa.

The protein (BSA) adsorption characteristic of a mono-layer of themacroporous gel-filled membrane was examined using the generalprocedures one for a single membrane disk and one for a multi-membranestack, as described above. The membrane stack contained 7 membranelayers of total thickness 1.75 mm. In both experiments the proteinconcentration was 0.4 g/L in a 50 mM TRIS buffer solution, and the flowrate of the protein solution used was 3.1±0.1 ml/min, delivered byperistaltic pump. The breakthrough capacity for BSA was 64 mg/ml in thesingle membrane experiment and 55±2 mg/ml in the multi-membrane stackexperiment.

Example 26

This example illustrates the use of a mixture of two monomers in makinga positively charged macroporous gel-filled membrane.

A 10 wt-% solution was prepared by dissolving diallyldimethylammoniumchloride (DADMAC) and (3-acrylamidopropyl)trimethylamonium chloride(APTAC), in the ratio 50:50, in a solvent mixture containing 65 wt-%dioxane, 18 wt-% water and 17 wt-% DMF. N,N′-methylenebisacrylamide(BIS) was added to the monomer solution to obtain 40% (mol/mol)cross-linking degree. The photoinitiator Irgacure® 2959 was added in theamount of 1% with respect to the total mass of the monomers.

A sample of the non-woven polypropylene substrate TR2611A was placed ona polyethylene sheet and filled with the monomer solution. The substratewas subsequently covered with another polyethylene sheet and theresulting sandwich was run between two rubber rollers to press themonomer solution into the pores and remove excess of solution. Thefilled substrate was irradiated at 350 nm for 20 min for thepolymerization process to occur. The macroporous gel-filled membrane wasremoved from between the polyethylene sheets, washed with water,TRIS-buffer solution and stored in water for 24 hrs.

The macroporous gel-filled membrane produced by this method had a waterflux in the range of 2550 kg/m² hr at 70 kPa. The mass gain determinedwith a duplicate sample was found to be 45%.

The protein (BSA) adsorption characteristic of the mono-layermacroporous gel-filled membrane was examined using the general procedurefor a single membrane disk described above. A solution of BSAconcentration of 0.4 g/L in a 50 mM TRIS buffer solution was deliveredto the membrane at a flow rate of 2-4 ml/min. The breakthrough capacityof the macroporous gel-filled membrane was 40 mg/ml.

Example 27

This example illustrates the effect of addition of a neutral monomer tothe mixture of charged monomers used in example 26.

A 15 wt-% solution was prepared by dissolving diallyldimethylammoniumchloride (DADMAC), (3-acrylamidopropyl)trimethylammonium chloride(APTAC), and acrylamide (AAM), which were taken in the ratio 40:40:20,in a solvent mixture containing of 65 wt-% dioxane, 17 wt-% of water,and 18 wt-% of DMF. N,N′-methylenebisacrylamide (BIS) was added to themonomer solution to obtain 20% (mol/mol) cross-linking degree. Thephotoinitiator Irgacure® 2959 was added in the amount of 1% with respectto the total mass of the monomers.

A sample of the non-woven polypropylene substrate TR2611A was placed ona polyethylene sheet and filled with the monomer solution. The substratewas subsequently covered with another polyethylene sheet and theresulting sandwich was run between two rubber rollers to press themonomer solution into the pores and remove excess of solution. Thefilled substrate was irradiated at 350 nm for 20 min for thepolymerization process to occur. The macroporous gel-filled membrane wasremoved from between the polyethylene sheets, washed with water,TRIS-buffer solution and stored in water for 24 hrs.

The macroporous gel-filled membrane produced by this method had a waterflux of 550 kg/m² hr at 70 kPa and a mass gain (determined using aduplicate samples) of 65 wt-%.

The protein (BSA) adsorption characteristic of the mono-layermacroporous gel-filled membrane was examined using the general procedurefor a single membrane disk described above. A solution of BSAconcentration of 0.4 g/L in a 50 mM TRIS buffer solution was deliveredto the membrane at a flow rate of 3.5-4 ml/min. The breakthroughcapacity of the macroporous gel-filled membrane was 130 mg/ml.

Example 28

This example illustrates the formation of unsupported positively chargedmacroporous gel by cross-linking of a preformed polymer.

A 10% solution of poly(allylamine hydrochloride) PAH was prepared bydissolving the polymer in a solvent mixture containing 60% water and 40%iso-propanol (2-propanol). The polymer was partially deprotonated (40%)by adding 6.67 N NaOH. Ethylene glycol diglycidyl ether (EDGE) was addedto this solution to obtain 40% (mol/mol) degree of cross-linking. Thesolution was kept at room temperature for 3 hours for gel formation bythe cross-linking reaction between the amine groups of PAH and epoxygroups of EDGE. After 3 hours, the gel was placed in a water bath forall un-reacted chemicals to leach out.

A sample of the wet gel was examined using ESEM. The micrograph shown inFIG. 22 indicates that a macroporous gel was formed with the porediameter of about 70-80 μm. The wet gel was mechanically very weak.

Example 29

This example illustrates the making macroporous gel incorporated in anon-woven fabric support.

A 10 wt-% solution of poly(allylamine hydrochloride) (PAH) was preparedas in Example 28. The polymer was partially deprotonated and EDGE addedas described in Example 25 and the solution was applied to a sample ofthe non-woven polypropylene membrane support TR2611A placed between twopolyethylene sheets. The resulting sandwich was run between two rubberrollers to press the polymer solution into the pores of the substrate,spread it evenly, and remove the excess solution. The solution-filledsupport sample was kept at room temperature for 3 hours forcross-linking process to take place resulting in the formation of gel.After that time, the macroporous gel-filled membrane was removed fromthe sandwich and placed in a water bath for 12 hours to leach outunreacted chemicals.

A wet sample of the resulting composite membrane was examined usingESEM. The micrograph shown in FIG. 23 indicates the composite membranehaving macroporous gel in the fibrous non-woven support member. Theaverage pore size of the gel was about 25-30 μm. The membrane thicknesswas 800 μm and the water flux measured at 100 kPa was 592 kg/m²h. Themacroporous gel-filled membrane showed rather low BSA binding capacityof about 10 mg/ml.

Example 30

This example provides a comparison of the protein adsorption by acomposite membrane with the commercial Mustang® Coin Q produced by PallCorporation.

A macroporous gel-filled membrane prepared in example 22 was tested in amulti-membrane stack of 7 membrane layers of a total thickness of 1.75mm, according to the testing protocol described in Example 25. AMustang® Coin Q was also tested under similar conditions. The membranestack was prepared by placing seven (7) layers of the membrane sample inthe wet state on top of each other. The assembled membrane stack waslightly compressed pressed to remove excess of water. The membrane stackwas then heated in an oven at 60-70° C. for at least 30 min. Thethickness of the resulting membrane stack in the dry state was 1.8-1.9mm. This process produced a stack in which the multiple membrane layersadhered to each other. The results shown in FIG. 24 indicate that bothsystems give similar performances.

Example 31

This example provides the hydrodynamic (Darcy) permeability of referencemacroporous gel-filled membranes containing porous support member andhomogeneous gels filling the pores of the support. The homogeneous gelswere obtained by using thermodynamically good solvents and theirhomogeneity was assessed based on transparency of simultaneously formedunsupported gels of the same composition. Clear and transparent gelswere assumed to be homogeneous, contrary to macroporous gels that werealways found opaque.

(A) Glycidyl Methacrylate Based Homogeneous Gel-Filled Composites

The macroporous gel-filled membranes containing homogeneous gels ofglycidyl methacrylate-co-ethylene glycol dimethacrylate, GMA-co-EDMA,were prepared using 1,4-dioxane as a solvent and 4.7 wt-% of EDMA(cross-linker) in monomer mixture, and different total monomerconcentrations. Poly(propylene) support PP1545-4 and the generalprocedure for preparing the macroporous gel-filled were used. DMPA wasused as photoinitiator and the irradiation was carried out at 350 nm for120 minutes. The hydrodynamic permeability of the membranes was measuredand an empirical equation was derived for the relationship between thehydrodynamic permeability, k, and the mass gain of the compositemembranes containing poly(GMA-co-EDMA) homogeneous gels in the PP1545-4support. The equation is as follows:k=3.62×10³ ×G ^(−9.09)

The differences between the measured values of permeability and thatcalculated from the above equation were found to be less than ±3%. Thisempirical relationship was subsequently used to calculate permeabilityof reference macroporous gel-filled membranes at different mass gains.

(B) Poly(diallyldimethylammonium chloride) Based Homogeneous Gel-FilledComposites

The macroporous gel-filled membranes containing homogeneous gels ofdiallyldimethylammonium chloride-co-methylenebisacrylamide,DADMAC-co-BIS, were prepared using water as a solvent and 1.0 wt-% ofBIS cross-linker in monomer mixture, and different total monomerconcentrations. Poly(propylene) support PP1545-4 was coated with TritonX-114 surfactant by immersing the support samples in 2t-% solution ofthe surfactant in methanol/water (60/40) mixture for 16 hours followedby drying in air. The general procedure for preparing the macroporousgel-filled membranes used to make homogeneous gel-filled membranes.Irgacure® 2959 was used as photoinitiator and the irradiation wascarried out at 350 nm for 30-40 minutes. The hydrodynamic permeabilityof the series of membranes was measured and an empirical equation wasderived for the relationship between the Darcy permeability, k, and themass gain, G:k=2.09×10⁻¹² ×G ^(−4.01)(C) Acrylamide Based Homogeneous Gel-Filled Composites

The hydrodynamic permeability of homogeneouspoly(acrylamide)-co-methylenebisacrylamide, AAM-co-BIS, was estimatedfrom the empirical relationship between the gel permeability and the gelpolymer volume fraction provided by Kapur et al. in Ind. Eng. Chem.Res., vol. 35 (1996) pp. 3179-3185. According to this equation, thehydrodynamic permeability of a poly(acrylamide) gel,k_(gel)=4.35×10⁻²²×Φ^(−3.34), where Φ is the polymer volume fraction inthe gel. In the same article, Kapur et al. provide a relationshipbetween the hydrodynamic permeability of a gel and a porous membranefilled with the same gel. According to this relationship, thepermeability of the membrane, k_(membrane)=(ε/τ)×k_(gel), where ε is theporosity of the support and τ is the tortuosity of the support pores.The pore tortuosity can be estimated as a ratio of the Kozeny constant,K, for a given porosity, i.e., K=5, and the Kozeny constant for astraight cylindrical capillary equal to 2. Thus, for the poly(propylene)support PP1545-4 with porosity of 0.85, the ratio (ε/τ)=0.85/2.5=0.34.

The polymer volume fraction, Φ, can be converted to mass gain using thepartial specific volume, ν₂, for poly(acrylamide) and the density, ρ, ofpoly(propylene). The values of these parameters can be found in PolymerHandbook, edited by Brandrup et al., Chapter VII, Wiley and Sons, NewYork, 1999. Thus, the mass gain of a macroporous gel-filled membranecontaining poly(propylene) support of porosity ε filled with a gel whosepolymer occupies the fraction Φ of the pores is given by

${{Mass}\mspace{14mu}{Gain}\mspace{14mu}(\%)} = {\frac{\varphi/v_{2}}{\left( {1 - ɛ} \right)\rho} \times 100\;\%}$

The above equation was combined with that of Kapur et al. to give anempirical relationship allowing one to calculate hydrodynamicpermeability of reference macroporous gel-filled membranes, k, atdifferent mass gains, G. The combined equation is as follows:k=1.80×10⁻¹² ×G ^(−3.34)

The equation is valid for ρ=0.91 g/cm³; ε=0.85; ν₂=0.7 cm³/g;(ε/τ)=0.34.

(D) Poly(AMPS) Based Homogeneous Gel-Filled Composites

The macroporous gel-filled membranes containing homogeneous gels of2-acrylamido-2-propane-1-sulfonic acid-co-methylenebisacrylamide,AMPS-co-BIS, were prepared using water as a solvent and 10.0 wt-% of BIScross-linker in monomer mixture, and different total monomerconcentrations. Poly(propylene) support PP1545-4 was coated with TritonX-114 surfactant by immersing the support samples in 2t-% solution ofthe surfactant in methanol/water (60/40) mixture for 16 hours followedby drying in air. The general procedure for preparing the macroporousgel-filled membranes was used to make homogeneous gel-filled membranes.Irgacure® 2959 was used as photoinitiator and the irradiation wascarried out at 350 nm for 60 minutes. The hydrodynamic permeability ofthe series of membranes was measured and an empirical equation wasderived for the relationship between the Darcy permeability, k, and themass gain, G:k=2.23×10⁻¹⁶ ×G ^(−1.38)(E) Poly(APTAC) Based Homogeneous Gel-Filled Composites

The macroporous gel-filled membranes containing homogeneous gels of(3-acrylamidopropane)trimethylammoniumchloride-co-methylenebisacrylamide, APTAC-co-BIS, were prepared usingwater as a solvent and 10.0 wt-% of BIS cross-linker in monomer mixture,and different total monomer concentrations. Poly(propylene) supportPP1545-4 was coated with Triton X-114 surfactant by immersing thesupport samples in 2t-% solution of the surfactant in methanol/water(60/40) mixture for 16 hours followed by drying in air. The generalprocedure for preparing the macroporous gel-filled membranes was used tomake homogeneous gel-filled membranes. Irgacure® 2959 was used asphotoinitiator and the irradiation was carried out at 350 nm for 60minutes. The hydrodynamic permeability of the series of membranes wasmeasured and an empirical equation was derived for the relationshipbetween the Darcy permeability, k, and the mass gain, G:k=9.51×10⁻¹⁶ ×G ^(−1.73)(F) Poly(ethyleneimine) Based Homogeneous Gel-Filled Composites

The macroporous gel-filled membranes containing homogeneous gels ofbranched poly(ethyleneimine) cross-linked with ethylene glycoldiglycidyl ether (EDGE) were prepared using methanol solutions of BPEIof different concentrations. The degree of cross-linking used was 10mol-%. Poly(propylene) support PP1545-4 was used together with thegeneral procedure of fabrication of macroporous gel-filled membranes byin situ cross-linking of cross-linkable polymers described in Example10. A series of membranes with different mass gains was prepared bychanging the concentration of PEI in the solution. The Darcypermeability of the membranes was measured and an empirical equationdescribing the relationship between the permeability, k, and the massgain, G, was derived. The equation is as follows:k=4.38×10⁻¹⁴ ×G ^(−2.49)

Example 32

This example provides comparison between Darcy permeability ofmacroporous gel-filled membranes containing supported macroporous gelsand the permeability of the reference membranes containing homogeneousgels filling the porous support member.

The comparison is shown in Table 10 below.

TABLE 10 Permeability Ratio Values for Gel-Filled Membranes DarcyPermeability Membranes containing Macroporous Gels of ReferencePermeability Darcy Permeability Membrane Ratio Example Massk_(macroporous) k_(homogeneous) k_(macroporous)/ No. Gain % m² m²k_(homogeneous)  5 107 9.53 × 10⁻¹⁶ 2.93 × 10⁻¹⁹ 3.2 × 10³  8  74 1.58 ×10⁻¹⁵ 2.49 × 10⁻¹⁸ 6.3 × 10²  9  92 * 1.98 × 10⁻¹⁸ * 1.85 × 10⁻¹⁸ * 1.1× 10⁰ * 100 3.53 × 10⁻¹⁶ 1.65 × 10⁻¹⁸ 2.2 × 10² 100 * 5.19 × 10⁻¹⁸ *1.65 × 10⁻¹⁸ * 3.2 × 10⁰ * 10 103  4.4 × 10⁻¹⁵ 1.58 × 10⁻¹⁸ 2.8 × 10³ 11 42 9.87 × 10⁻¹⁶ 8.81 × 10⁻¹⁹ 1.1 × 10³ 12  80 1.09 × 10⁻¹⁶ 6.78 × 10⁻²⁰1.6 × 10³ 13  95 1.89 × 10⁻¹⁵ 5.40 × 10⁻¹⁹ 3.5 × 10³ 14 144 2.77 × 10⁻¹⁵8.39 × 10⁻¹⁹ 3.3 × 10¹ 158 9.15 × 10⁻¹⁶ 3.77 × 10⁻¹⁷ 2.4 × 10¹ 237 1.66× 10⁻¹⁷ 9.20 × 10⁻¹⁹ 1.8 × 10¹ 265 2.48 × 10⁻¹⁵ 3.34 × 10⁻¹⁹ 7.5 × 10³277 6.96 × 10⁻¹⁶ 2.24 × 10⁻¹⁹ 3.1 × 10³ * denotes homogeneous ormicro-heterogeneous gels in membranes (Comparative) Darcy PermeabilityMembraness containing Macroporous Gels of Reference Permeability DarcyPermeability Membrane Ratio Example Mass k_(macroporous) k_(homogeneous)k_(macroporous)/ No. Gain % m² m² k_(homogeneous) 15 103 1.59 × 10⁻¹⁸ *3.38 × 10⁻¹⁹ * 4.7 × 10⁰ * 108 2.52 × 10⁻¹⁸ * 2.93 × 10⁻¹⁹ * 8.6 × 10⁰ *110 2.11 × 10⁻¹⁸ * 2.70 × 10⁻¹⁹ * 7.8 × 10⁰ * 112 9.32 × 10⁻¹⁸ 2.61 ×10⁻¹⁹ 3.6 × 10¹ 130 5.34 × 10⁻¹⁶ 1.56 × 10⁻¹⁹ 3.4 × 10³ 273 8.57 × 10⁻¹⁷1.31 × 10⁻²⁰ 6.5 × 10³ 307 3.88 × 10⁻¹⁷ 8.87 × 10⁻²¹ 4.4 × 10³ 18 1132.26 × 10⁻¹⁶ 1.39 × 10⁻¹⁸ 1.6 × 10² 19  51 3.73 × 10⁻¹⁵ 4.16 × 10⁻¹⁸ 9.0× 10² 20  34 7.52 × 10⁻¹⁵ 7.18 × 10⁻¹⁸ 1.0 × 10³ 21 108 6.47 × 10⁻¹⁶1.47 × 10⁻¹⁸ 4.4 × 10² 22  46 4.52 × 10⁻¹⁵ 4.85 × 10⁻¹⁸ 9.3 × 10² *membranes.

Examples 33-40

These examples illustrate a method of preparing a responsive macroporousgel-filled membrane using photoinitiated free radical polymerization ofacrylic acid (AA) (ionic monomer), acrylamide (AA), andtrimethylolpropane triacrylate (TRIM) as a cross-linker. The molar ratioof acrylic acid to acrylamide was 1:1 and 1,4-dioxane was used as asolvent in all experiments. Monomer solution compositions andpolymerization conditions are given in Table 11. After polymerization,the responsive macroporous gel-filled membrane was washed withde-ionized water for about 16 hrs.

TABLE 11 Monomer solution compositions and polymerization conditionsTotal Concentration of Monomer Degree of Irradiation Example SampleSupport Mixture Cross-linking Time Mass Gain no. ID Member (wt-%)(mol-%) (min) (%) 33 AM675 TR2611A 19.9 5.0 20 115.6 34 AM678 TR2611A13.3 5.0 90 81.7 35 AM680 TR2611A 12.8 5.2 20 82.3 36 AM681 TR2611A 12.610.8 15 88.8 37 AM682 TR2611A 23.8 10.9 15 167.3 38 AM684 TR2611A 31.010.8 10 217.3 39 AM683 TR2611A 38.8 10.8 15 294.6 40 AM694 PP 1545-424.0 10.2 10 218.0

The amount of gel formed in the support member depends on the porevolume available to fill, the total concentration of the monomermixture, and the degree of conversion in the polymerization. In FIG. 25,the mass gain obtained with support TR2611A is plotted as a function oftotal monomer concentration. The data can be approximated to fall withina straight line (R²=0.97), indicating a similar degree of conversion foreach sample. The experimental values are very close to their theoreticalcounterparts estimated from the pore volume in the support and themonomer concentration. This suggests that the degree of conversion isclose to 100% and that an irradiation time of 10 minutes is sufficientunder the light conditions applied. As expected, the mass gain obtainedwith the PP 1545-4 support was higher than that obtained with theTR2611A support due to the larger porosity of the former (85 vol-%versus 79.5 vol-%).

Example 41

This example illustrates the responsiveness of the responsivemacroporous gel-filled membranes according to Example 33 to ionicinteractions. For this purpose, the macroporous gel-filled membraneswere tested with solutions of different pH and/or salt concentrations bymeasuring the flux at 100 kPa. A typical change in flux taking placewith the change of pH from about 3 (1 mM HCl) to about 12 (1 mM NaOH),obtained with membrane AM675 is shown in FIG. 26. It can be seen fromthe Figure that the flux measured with 1 mM HCl is nearly 100 timeslarger than the flux measured with 1 mM NaOH. The reason for thisbehavior of the membranes lies in changes in the degree of ionization ofthe acid component of the macroporous gel. At high pH (1 mM NaOH) thecarboxyl groups of the acid component become ionized and theelectrostatic repulsive force causes the polymer chains to uncoil andstretch until balanced by counteracting forces of the polymer networkelasticity and confinement imposed by the support member of themembrane. The swelling polymer chains reduce the pore volume and thepore radius in the gel. At low pH (1 mM HCl), the carboxyl groups areconverted into neutral carboxylic acid groups, the electrostatic forcesdisappear, and the gel shrinks (collapses) enlarging the pores in thegel. The presence of the support member prevents the gel from collapsingas a whole, i.e., from the process that would occur in the unsupportedbulk gel, and closing the pores. Thus, the presence of the supportreverses the direction in which hydraulic properties of the gel change.When pure water flux is measured, the values obtained depend on thedistance from equilibrium ionization at the water pH (˜5.5). The initialwater flux can be assumed to be measured at equilibrium. Immediatelyafter the acid or base, the gel is far from equilibrium with water andthe pure water flux reflects this state by being close to the flux inthe ionized form (after NaOH) or neutral form (after HCl).

The ratio of the flux measured with 1 mM HCl to that measured with 1 mMNaOH has been taken as a measure of membrane response (MR). The resultsobtained with membranes described in Example 33-40 are shown in Table12.

TABLE 12 Results for Examples 33-40 Membrane ID AM675 AM678 AM680 AM681AM682 AM684 AM683 AM694 Total Monomer 19.9 13.3 12.8 12.6 23.8 31.0 38.824.0 Conc. Wt-% Degree of 5.0 5.0 5.2 10.8 10.9 10.8 10.8 10.2Cross-linking Mol-% Membrane 89.6 372.2 352.0 29.4 10.3 5.6 4.8 19.5Response (MR)

The results in Table 12 show that the response of the compositemembranes to ionic interaction can also be controlled by the totalconcentration of monomer mixture and the degree of cross-linking. As themonomer concentration increases, the membrane sensitivity to theenvironmental changes decreases. Similar effect is found when the degreeof cross-linking is increased.

Example 42

This example illustrates the ability of membranes based on responsivemacroporous gel-filled membranes to fractionate proteins. The separationof therapeutic proteins Human Serum Albumin (HSA) and HumanImmunoglobulin G (HIgG) was chosen as a case study. Human plasma is thestarting material for the production of a number of therapeuticproteins, which are referred to as plasma proteins. The most abundantamongst these are HSA and HIgG, both of which are manufactured in bulkquantities. These proteins are generally fractionated by precipitationbased processes which give high product throughput but poor resolutionin terms of separation. Membrane based processes such asultrafiltrations have the potential for giving both high throughput andhigh resolution.

Two composite membranes containing responsive macroporous gel,duplicates of membrane AM695 (see Tables 11 and 12), were tested fortheir suitability in separation of these plasma proteins. In theexperiments discussed here, the change in membrane pore size with changein salt concentration was utilized to effect protein-protein separationin the manner desirable, i.e. sequential release from the membranemodule. Other environmental conditions such as pH could well be used toachieve a similar objective.

A binary carrier phase system was used in the ultrafiltrationexperiments. The starting carrier phase in all the experiments was onewith a low salt concentration (typically 5-10 mM NaCl). In all theexperiments the carried phase was switched to one with a high saltconcentration (typically 1 M NaCl). The change in salt concentrationwithin the membrane module could be tracked by observing theconductivity of the permeate stream. The change in transmembranepressure gave an idea about the change in membrane hydraulicpermeability with change in salt concentration. FIG. 27 shows thechanges of transmembrane pressure and conductivity as a function of thepermeate salt concentration (FIGS. 27. A and B) and the changes oftransmembrane pressure as a function of permeate conductivity (FIG.27C). In this experiment the salt concentration was being increasedcontinuously in a linear fashion. The transmembrane pressure observed isrelated to the permeate salt concentration and reflects changes inpore-diameter.

Experiments were carried out using human serum albumin and humanimmunoglobulin mixtures. The ultrafiltration was started at a low saltconcentration (i.e. 10 mM). At this condition, human serum albumin wastransmitted while human immunoglobulin G was almost totally retained.The salt concentration was then increased and this increased the porediameter (as evident from drop in pressure in constant permeate fluxultrafiltration). This in turn led to the transmission of humanimmunoglobulin G through the membrane. Hence by altering theenvironmental condition it was possible to sequentially transmitproteins having different sizes through the same membrane. If theinitial mixture had also contained a protein significantly larger thanhuman immunoglobulin, it would have been possible to fractionate thethree proteins (i.e. human serum albumin, human immunoglobulin and thesignificantly bigger protein) by appropriately controlling the change insalt concentration. Two of the three fractions obtained here would be inthe permeate while the third fraction would be in the retentate.

The results obtained with duplicates of membrane AM694 are shown inFIGS. 28, 29, and 30. FIG. 28 shows the results obtained with HIgGultrafiltration. As evident from the figure, very little, if any HIgGwas transmitted at the low salt concentration. However, when the saltconcentration was increased, the HIgG was released from the membranemodule. The drop in TMP with increase in salt concentration was due tothe increase in pore diameter.

The results presented in FIG. 29 were obtained with HSA ultrafiltration.As evident from the figure HSA was freely transmitted through themembrane even at low salt concentration. When the salt concentration wasincreased, the transmission of HSA was found to increase a bit.

FIG. 30 shows the results obtained with HSA/HIgG ultrafiltration. At lowsalt concentration, HSA alone was transmitted. Ultrafiltration wascontinued until HSA was nearly completely removed from the membranemodule. The HIgG was then released by increasing the salt concentration.

Example 43

This example illustrates a method for making a positively chargedmacroporous gel-filled membrane having a high protein binding capacity.

A 15 wt-% solution was prepared by dissolving(3-acrylamidopropyl)-trimethylammonium chloride (APTAC),N-(hydroxymethyl)acrylamide and N,N′-methylenebisacrylamide ascross-linker in a molar ratio of 1:0.32:0.1, respectively, in a solventmixture containing 10 wt-% water, 60 wt-% di(propylene glycol)methylether and 30 wt-% dimethylformamide (DMF). The photo-initiator Irgacure®2959 was added in the amount of 1% with respect to the mass of themonomers.

A sample of the fibrous non-woven polypropylene substrate TR2611A wasplaced on a polyethylene sheet and filled with the monomer solution. Thesubstrate was subsequently covered with another polyethylene sheet andthe resulting sandwich was run between two rubber rollers to press themonomer solution into the pores and to remove any excess solution. Thesubstrate was irradiated for 5 minutes at 350 nm. The macroporousgel-filled membrane was then removed from between the polyethylenesheets, washed with water and TRIS-buffer solution and stored in waterfor 24 hrs.

Several samples were prepared according to the above process, and thesamples were then dried and weighed. The average mass gain of themacroporous gel-filled membrane was 55.7% of the original weight of thestarting support member.

The protein (BSA) adsorption characteristic of a multi-membrane stack ofthe above macroporous gel-filled membrane was examined using the generalprocedure for a mono-layer of the macroporous gel-filled membrane, asdescribed earlier. The membrane stack tested contained 4 membranelayers, giving a total thickness 1.05 mm. The protein solution used wasa 25 mM TRIS buffer solution with a protein concentration of 0.4 g/L,and the flow rate of the protein solution was 5.0 ml/min at 150 kPa. Thebreakthrough capacity for BSA was 281 mg/ml. In a subsequent desorptionstep, approximately 85% of the BSA was recovered.

All references mentioned herein are incorporated herein by reference tothe same extent as if each reference were stated to be specificallyincorporated herein by reference.

To those skilled in the art, it is to be understood that many changes,modifications and variations could be made without departing from thespirit and scope of the present invention as claimed hereinafter.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A membrane stack, comprising: first and second membrane layers; and aspacer layer disposed between said first and second membrane layers;wherein said membrane stack is configured such that fluid passes throughsaid membrane stack in a direction substantially perpendicular to theplane of said membrane layers and said spacer layer; at least one ofsaid membrane layers comprises a macroporous gel-filled membrane layer;the macroporous gel-filled membrane layer comprises a support member;the support member comprises a plurality of pores; said pores aresubstantially completely occupied by a macroporous gel; said pores areof average size between about 0.1 μm and about 25 μm; the macroporousgel has macropores of average size between about 10 and about 3000 nm;and the macropores of the macroporous gel are smaller than the pores ofthe support member.
 2. The membrane stack according to claim 1,comprising at least three membrane layers and at least two spacerlayers.
 3. The membrane stack according to claim 1, comprising at leastfive membrane layers and at least four spacer layers.
 4. The membranestack according to claim 1, comprising at least ten membrane layers andat least nine spacer layers.
 5. The membrane stack according to claim 1,wherein the macroporous gel-filled membrane layer comprises a responsivemacroporous cross-linked gel.
 6. The membrane stack according to claim5, wherein the responsive macroporous cross-linked gel is responsive tovariations in at least one of pH, ionic strength, temperature, lightintensity, or electrochemical current.
 7. The membrane stack accordingto claim 1, comprising at least one spacer layer that is formed of amesh.
 8. The membrane stack according to claim 1, wherein said spacerlayer has a thickness of about 50 μm to about 500 μm.
 9. The membranestack according to claim 1, wherein said spacer layer has openingshaving an average diameter of about 50 μm to about 5000 μm.
 10. Themembrane stack according to claim 1, further comprising a layer that isnot a membrane layer or a spacer layer.
 11. The membrane stack accordingto claim 1, comprising at least one membrane layer that is amicrofiltration membrane layer.
 12. The membrane stack according toclaim 1, comprising at least one membrane layer that is anultrafiltration membrane layer.
 13. The membrane stack according toclaim 1, comprising at least one membrane layer that is an affinity typemembrane layer.
 14. The membrane stack according to claim 1, whereinsaid membrane layers and said spacer layers are interleaved so that nomembrane layer is immediately adjacent to another membrane layer and nospacer layer is immediately adjacent to another spacer layer.
 15. Amodule comprising a membrane stack, wherein said membrane stackcomprises: first and second membrane layers; and a spacer layer disposedbetween said first and second membrane layers; said membrane stack isconfigured such that fluid passes through said membrane stack in adirection substantially perpendicular to the plane of said membranelayers and said spacer layer; at least one of said membrane layerscomprises a macroporous gel-filled membrane layer; said macroporousgel-filled membrane layer comprises a support member; said supportmember comprises a plurality of pores; said pores are substantiallycompletely occupied by a macroporous gel; said pores are of average sizebetween about 0.1 μm and about 25 μm; the macroporous gel has macroporesof average size between about 10 and about 3000 nm; the macropores ofthe macroporous gel are smaller than the pores of the support member;and said module has a fluid flow path that is substantiallyperpendicular to the plane of the major surface of the membrane andspacer layers in said membrane stack.
 16. The module according to claim15, wherein said membrane stack is in a flat configuration clamped orsealed at the edges, or in a spiral wound, or tubular configuration. 17.A method for separating a substance from a fluid, comprising the step ofpassing said fluid through a membrane stack, wherein said membrane stackcomprises first and second membrane layers; and a spacer layer disposedbetween said first and second membrane layers; said membrane stack isconfigured such that fluid passes through said membrane stack in adirection substantially perpendicular to the plane of said membranelayers and said spacer layer; at least one of said membrane layerscomprises a macroporous gel-filled membrane layer; said macroporousgel-filled membrane layer comprises a support member; said supportmember comprises a plurality of pores; said pores are substantiallycompletely occupied by a macroporous gel; said pores are of average sizebetween about 0.1 μm and about 25 μm; the macroporous gel has macroporesof average size between about 10 and about 3000 nm; and the macroporesof the macroporous gel are smaller than the pores of the support member.18. The membrane stack according to claim 1, wherein said spacer layerhas openings having an average diameter of about 500 μm to about 1000μm.
 19. A method for separating a substance from a fluid, comprising thestep of passing said fluid through a module, wherein said modulecomprises a membrane stack; said membrane stack comprises: first andsecond membrane layers; and a spacer layer disposed between said firstand second membrane layers; said membrane stack is configured such thatfluid passes through said membrane stack in a direction substantiallyperpendicular to the plane of said membrane layers and said spacerlayer; at least one of said membrane layers comprises a macroporousgel-filled membrane layer; said macroporous gel-filled membrane layercomprises a support member; said support member comprises a plurality ofpores; said pores are substantially completely occupied by a macroporousgel; said pores are of average size between about 0.1 μm and about 25μm; the macroporous gel has macropores of average size between about 10and about 3000 nm; the macropores of the macroporous gel are smallerthan the pores of the support member; and said module has a fluid flowpath that is substantially perpendicular to the plane of the majorsurface of the membrane and spacer layers in said membrane stack.